Apoptosis-regulating proteins

ABSTRACT

Adenovirus E1B 19 kD protein protects against cell death induced by viral infection and certain external physical and chemical stimuli. Activity of the 19 kD protein is similar to the cell death suppressing activity of the protein coded by the Bcl-2 protooncogene. Bcl-2 protein can functionally substitute for the E1B 19 kD protein during adenovirus infection and in transformation of primary cells with adenovirus E1A. Five different cDNA&#39;s for proteins, designated Nip1, Nip2, Nip3, Bip1A and Bip13 that specifically interact with the 19 kD protein were found. Mutational analysis of the interaction indicates that at least four of the proteins (Nip1, Nip2, Nip3 and Bip1A) associate with 19 kD protein at specific sites thereof. Homologous motifs are found on Bcl2. An additional protein, (Bip5), interacts with Bcl-2 but not with the 19K protein.

This invention was made with government support under grant number CA-33616 awarded by the National Institutes of Health-National Cancer Institute. The government has certain rights in the invention.

This is a Continuation of application Ser. No. 08/284,139 filed 2 Aug. 1994, abandoned.

BACKGROUND OF THE INVENTION

Apoptosis is a naturally occurring cell death characterized by reduction in cell volume, nuclear condensation, cell blebbing, and endonucleolytic degradation of DNA at nucleosomal intervals. The onset of apoptosis arises from an activation of one or more genetic programs. Apoptosis is distinct from cell necrosis where chromatin clumps, organelles swell and eventually the cell membrane dissolves (Edgington, 1993).

The 19 kD (19K) protein coded by the adenovirus (Ad) E1B region confers a survival function in adenovirus-infected cells and prevents premature cell death. The DNA fragmentation observed in cells infected with E1B 19K mutants is reminiscent of that observed during apoptosis (Wyllie, 1980). Hence, one can infer that the 19 kD protein protects against a cell death program induced by viral infection, thus facilitating efficient virus replication. The E1A proteins, and specifically the conserved region (CR) 1 and CR2 which interact with cellular proteins p300 and pRb and induce cellular proliferation, have been implicated in the onset of the virus-induced death program (White et al., 1991; Mymryk et al., 1994).

The 19 kD protein suppresses the cytotoxic effects of certain external stimuli such as tumor necrosis factor (TNF)-α (Gooding et al., 1991; White et al., 1992) and anti-Fas antibody (Hashimoto et al., 1991). Both TNF-α and anti-Fas have been shown to cause cell death through apoptosis (Laster et al., 1988; Itoh et al., 1991; Watanabe-Fukunaga et al., 1992). Similarly, the 19 kD protein protects cells against the effects of DNA damaging agents such as the anti-cancer drug cisplatin (Subramanian et al., 1993) and UV (Tarodi et al., 1993). Both cisplatin (Sorenson & Eastman, 1988) and UV (reviewed by Williams, 1991) induce cell death through the apoptotic pathway.

Levels of p53 increase in response to DNA damaging agents and it appears that the accumulation of p53 is responsible for the induction of apoptosis by DNA damaging agents (Clarke et al., 1993; Lowe et al., 1993; Lane, 1993). Since 19K can efficiently suppress cell death induced by DNA damaging agents (Subramanian et al., 1993; Tarodi et al., 1993), it appears that 19K suppresses p53-dependent apoptosis (Debbas and White, 1993). Thus, the 19 kD protein provides a survival function in virus-infected cells and also protects cells against certain other cell death-inducing stimuli.

The survival function provided by E1B 19K appears to be strikingly similar to the activity of the cellular proto-oncogene, bcl-2. The bcl-2 oncogene enhances the survival of hematopoietic B and T cells by blocking apoptosis (Vaux et al., 1988; Sentman et al., 1991; Strasser et al., 1991). Overexpression of Bcl-2 protein inhibits apoptosis induced by treatment with glucocorticoids (Alnemri et al., 1992a), deprivation of cytokines (Nunez et al., 1990; Hockenbery et al., 1990), activation of the c-myc oncogene (Bissonnette et al., 1992; Fanidi et al., 1992), radiation (Strasser et al., 1991) and other DNA damaging agents (Tarodi et al., 1993).

Thus, Bcl-2 appears to protect against apoptosis induced by diverse agents. The effect of the 19 kD protein on cell death induced by such a multitude of stimuli has yet to be examined. However, it is known that the Bcl-2 protein can substitute for the 19 kD protein during adenovirus infection. The characteristic fragmentation of cellular DNA induced by infection with Ad2 19K mutants is suppressed in cells expressing the human Bcl-2 protein (Tarodi et al., 1993). Similarly, expression of Bcl-2 by an adenovirus 2/Bcl-2 recombinant virus does not induce DNA fragmentation in infected cells and forms small plaques on cell monolayers. Rao et al. (1992) reported that Bcl-2 can substitute for 19K, albeit at reduced levels, in transformation of primary rat kidney cells in cooperation with E1A.

Studies have indicated that there are a number of molecules with homology to and that have similar activities with bcl-2. Hence, there might be termed an assemblage of functionally related molecules which includes BHRF-1, bcl-X_(L), bcl-X_(s), mcl-1 and the like. The similarity can extend to a structural homology at the nucleic acid level, protein level or both. But as will be revealed hereinbelow, the homology need not be over the length of the molecule but can be confined to discrete functional portions of the molecule. Hence, under that criterion, 19K also comprises the assemblage of molecules relating to apoptosis.

The mechanism by which the 19K gene and the Bcl-2 protooncogene protect against cell death is not known. It is possible that those apoptosis-regulating proteins (hereinafter blocking proteins) mediate cell survival by interacting with certain cellular apoptosis-regulating proteins. Identification of any such cellular apoptosis-regulating proteins which interact with E1B 19 kD protein and with Bcl-2 would enable regulation of apoptosis in cells.

SUMMARY OF THE INVENTION

An object of the instant invention is to provide proteins, and the nucleic acids encoding said proteins, which bind to proteins known to regulate cell survival.

Another object of the instant invention is to provide oligopeptides, and the nucleic acids encoding said oligopeptides which mediate the interaction between proteins of the instant invention and proteins known to regulate cell survival.

Yet another object of the instant invention is use of said proteins and oligopeptides which bind to proteins known to regulate cell survival in diagnostic assays to assess presence and activity of said cell survival regulating proteins.

Those and other objects have been achieved in the identification of a series of proteins which bind to the E1B 19K and Bcl-2 proteins, and in the identification of consensus sequences shared by the proteins which mediate the binding therebetween.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the functional organization of the 19 kD protein. The 19 kD sequences involved in suppression of cisplatin-induced cell death and E1A-cooperative transformation are based on Subramanian et al. (1993). In the top half of the figure, solid line bars indicate strong effects of various mutants on the 19 kD functions and interaction with cellular proteins, bars with dashed lines indicate weak effects. The sequence similarity between the 19 kD and Bcl-2 proteins and the mutants that map within those sequences (set off in boxes) are shown at the bottom of the figure (residues 50-56 of SEQ ID NO:8, residues 90-98 of SEQ ID NO:8, residues 106-112 of SEQ ID NO:16 and residues 43-51 of SEQ ID NO:16, respectively). The human Bcl-2 protein is a mutant form isolated from a human lymphoma and contains a Phe (instead of Ile) residue at position 48 (Seto et al. 1988, Hockenbery et al., 1990) and two other mutations (Seto et al., 1988). Identical amino acids are indicated by solid lines, similar amino acids by colons and distantly related amino acids by single dots.

FIGS. 2A, 2B and 2C depict sequence homologies. FIG. 2A Homology between Nip1 (amino acids 43-100 of SEQ ID NO:21) and the catalytic domain of rat oalmodulin (CaM)-dependent phosphodiesterase (PDE) (SEQ ID NO:34) (Repaske et al., 1992). Similar homologies are also observed with mouse and cattle PDE's.

FIG. 2B Putative Ca²⁻ -binding motif of Nip2 (amino acids 79-90 of SEQ ID NO:23). O indicates an oxygen-containing residue, * indicates non-conserved residues and underlined residues are those that conform to the consensus.

FIG. 2C Homology between Nip2 (amino acids 168-299 of SEQ ID NO:23) (lower case letters) and human GTPase activating protein, RhoGAP (SEQ ID NO:35), Barfod et al., 1993; Lancaster et al., 1994).

DETAILED DESCRIPTION OF THE INVENTION

Isolated, in the context of the instant invention, indicates that some intervention occurs which increases the level of purity of a molecule over that found in nature.

Derivatives in the context of the instant invention is contemplated to include any modifications to the molecules of interest, including truncation, fusion, complexing to carriers and the like which brings about a physical change in the parent molecule but does not alter substantially the biologic function thereof in relationship to the parent molecule. Biologic function may be enhanced or may be lowered so long as a desirable and tolerable level is maintained. A lowered activity is palatable when a subsidiary characteristic is enhanced, for example, solubility in an aqueous medium. Alternatively, a change in the sequence that gives a mutant may yield activity that is opposite of that of the parent molecule.

It should be noted that sequence identity whether at the peptide or nucleic acid level, while preferred under certain circumstances, is not a sine qua non condition in the instant invention. A key determinant, thus, is not the absolute sequence per se but the function ascribed to the molecule or to a product of the molecule. Hence, functional equivalence is the guiding directive as to the molecules of the instant invention.

Accordingly, by way of example, any one or all of the amino acids in the motifs described herein can be substituted so long as binding activity with the target of the parent sequence is retained. As noted hereinabove, as to the level of binding, that degree is mandated by the design, choice and end use of the motif derivative, and the artisan can configure and determine same without undue experimentation practicing the methods set forth herein.

The instant active agents regulate cell survival, that is, cell survival can be enhanced or prolonged or cell survival can be curtailed or shortened.

As noted hereinabove, there are molecules which have the effect of prolonging cell survival. Examples are Bcl-2 and E1B 19K. Such proteins appear to subvert apoptosis and thereby prolong cell survival. To prolong cell survival is to generate cells that statistically survive longer than expected.

The in vivo genetic strategy designated `two hybrid` cloning (Fields and Song, 1989; Chien et al., 1991) permits rapid genetic screening in yeast of molecules that associate and the method has been used to isolate from expression libraries cDNA clones that code for proteins interacting with several known proteins.

Briefly, the method relies on the double transformation of yeast hosts with plasmids that encode fusion proteins. One plasmid carries partial sequences for a reporter molecule, for example, the GAL4 DNA binding domain, at the amino terminus of the fusion protein and sequences for the known protein, to which a ligand is sought, also known as the "bait" at the carboxyl terminus. For example, the bait can be the 19K protein or Bcl-2 protein.

The second plasmid comprises sequences encoding a complementary protein for the reporter molecule, in the above case, required by the GAL4 DNA binding domain, such as the GAL4 activation domain, at the amino terminus and expressed products of individual cDNA's from a bank at the carboxyl terminus. A suitable host is used to enable the selection planned, in the scenario discussed, the host would be one wherein the expression of β-galactosidase is under the control of the GAL1 promoter.

Selection of double transformants are those that express β-galactosidase, hence would be blue colonies on an X-gal plate because the bait and protein encoded by the cDNA of the second plasmid bind and that interaction juxtaposes the two GAL4 regulatory elements required for β-galactosidase expression.

In the case where 19K or Bcl-2 is the bait, the cDNA's identified thereby, encode polypeptides that bind 19K, Bcl-2 or both. Hence, those proteins have a key role in apoptosis whether by functionally and directly regulating one or more of the observable characteristics (symptoms) of apoptosis or by affecting the survival enhancing activity of 19K or Bcl-2, possibly by binding thereto.

As is known in many biologic systems, because of the tertiary and quaternary configuration of molecules and the interactions therebetween, often small portions of a molecule can have key functions, such as a recognition function, a signalling function, a catalytic function, a binding function and the like. For example, a specific antigenic determinant may comprise as few as five to six contiguous amino acids of a polypeptide and the variability and diversity of antibodies is generated by a non-contiguous plurality of short amino acid stretches in the heavy and light chains that vary from clone to clone.

Similarly, the binding interaction of an apoptosis-regulating protein, such as 19K and bcl-2, with another apoptosis-regulating molecule, such as Nip1, Nip2, Nip3 and Bip1A, disclosed herein, is governed and mediated at least in part by certain sites of the proteins which are contiguous portions of the proteins which comprise short conserved motifs or consensus sequences of amino acids. The motifs can comprise 15 amino acids or less, often 10 amino acids or less. Two motifs are AAPAPGFFS (SEQ ID NO:1) and YRRDFAE (SEQ ID NO:2) of Bcl-2 and YKWEFEE (SEQ ID NO:3) and AAAAVAFLS (SEQ ID NO:4) of 19K. Those sequences play crucial roles in the interaction between regulating molecules. Related sequences, e.g., YbxaFxE (SEQ ID NO:5), where b is a basic amino acid, x is any amino acid and a is an acidic amino acid, and AAhAhFhS (SEQ ID NO:6), where h is a hydrophobic amino acid, may be important for the interaction as well.

It is likely that mutation at the site of the motif can have consequences related to the normal functioning of the apoptosis regulating molecules and interaction therebetween. For example, the motif from a normal B cell clone of Bcl-2 was found to be AAPAPGIFS (SEQ ID NO:7) (Cleary, 1986). On the other hand, the motif obtained from a follicular lymphoma-derived clone of bcl-2 was found to be AAPAPGFFS (SEQ ID NO:1). Also, the bcl-2 consensus may endure some variability having a sequence YRGDFAE (SEQ ID NO:32) (Tsujimoto & Croce, 1986).

That observation demonstrates the importance of the consensus sequence in the normal state and provides a differential marker for identifying and screening abnormal cells based on the amino acid substitution.

The instant nucleic acids and polypeptides may be obtained as described herein, that is by recombinant means, or may be used to obtain homologous nucleic acids and proteins by hybridization, for example, an instant nucleic acid can be used as a probe of a gene bank to identify clones with suitable homology therewith. Also, within the confines of available technology. The nucleic acids and polypeptides may be synthesized in vitro using, for example, solid phase oligonucleotide and oligopeptide synthetic methods known in the art.

The interactions between the apoptosis-regulating proteins, such as 19K and Bcl-2, and the proteins of the instant invention that bind thereto may take a variety of forms. For example, the molecules may either promote or suppress apoptosis on their own. The 19K and Bcl-2 type proteins may recruit the proteins of the instant invention and promote cell survival by enhancing the activity thereof or by suppressing the activity thereof.

The availability of ready sources of apoptosis-regulating proteins enables the manipulation of cells for a desired purpose. For example, it is believed that certain malignancies arise from over-expression of an apoptosis-blocking gene, such as bcl-2. In that case, application or administration of an instant protein, or nucleic acid encoding said protein, can counteract the high levels of Bcl-2. In the converse, application or administration of a protein such as Bcl-2 will enhance the life span of a cell.

Hence, the instant proteins find utility in the growth and maintenance of cells and tissues in culture. The instant proteins can be added to the culture medium in similar fashion to those supplements now used, such as, fetal calf serum, glutamine and the like. Whether an inducing or blocking molecule is used will depend on the needs of the investigator and the cell or tissue cultured.

The instant proteins that enhance cell survival can be used to maintain valuable cells without the usual "malignant" transformation methods now used. Hence, culture medium can contain one or more of the instant proteins or cells can be transfected by an expressable nucleic acid encoding the desired protein(s). Such a transfection can be by known means, such as microinjection or by precipitation, and the vector can be one which is retained episomally or integrates into the host genome. The vector comprises known regulatory elements, including optionally a signal sequence nucleic acid fragment, to ensure proper expression, such as a promoter, enhancer, terminator and the like. The starting materials and methods are available in the art.

It is evident that the instant molecules can find utility in vivo using those administration means found suitable for oligopeptides, proteins and nucleic acids. The instant molecules may be encapsulated, for example, in a liposome or in an implantable depot to overcome some of the known administration problems which might confound adequate delivery and activity at a site. Moreover, the instant molecules may be conjugated to cell binding molecules for direct targeting to a site.

The uses of the instant molecule are contigent on the impact thereof on cell survival. The use of biologics in pharmaceutic preparations is known in the art. Hence, the instant molecules could find application in treatment and amelioration of immune disorders, abnormal states characterized by aberrant cell growth and the like.

The instant molecules of apoptosis-regulating activity find utility in diagnostic assays for the presence and expression of relevant nucleic acids, presence and expression of relevant proteins, presence and functionality of binding between the two classes of molecules and the like. Use of, for example, the oligopeptide motifs in a solid-phase microassay would enable identifying whether functional apoptosis-regulating molecules are present in a sample. Hence, expression and extracellular presence of apoptosis-regulating proteins in degenerative diseases, such as Alzheimer's disease and spina bifida, may be diagnositic of those disorders.

Hence, the instant motif oligopeptides can be affixed to a solid phase, such as plastic microtiter dishes, using methods obtained from immunology for the fixing of antigen or antibody to a plate and then a sample is added. Following suitable washes, the presence of and amount of bound material is assessed, for example using a labelled antibody specific for the apoptosis-regulating molecules and the amount of label is assessed. Alternatively, the assessment can be visual under a microscope and the like. Alternatively, the motif can be labelled, applied to a sample and the label present assessed, similar to known radioreceptor assays, such as those for hormones using the hormone receptor as a specific reporter.

Such an assay which turns on the functional association between an apoptosis regulating molecule can be beneficial in identifying drugs that impede or prevent the interaction of the apoptosis regulating molecules.

As disclosed herein, some of the instant proteins bear homology to known proteins with a characteristic biologic activity. Hence, presence and amount of an apoptosis-regulating molecule can be ascertained by assaying for that biologic activity. For example, if a regulating molecule has enzymatic activity, then an assay for the presence and amount of that regulating molecule may rely on the assay of that enzymatic activity.

Hence, the amount of apoptosis-regulating molecules now can be ascertained in a sample using either of the two binding molecules as a receptor for the other.

Alternatively, rather than monitoring presence of the relevant molecules by physical characterization, for example, electrophoresis, or by activity, for example, an assay of an enzymatic activity, the instant molecules can be ascertained indirectly by ligands other than one or the other, for example, a ligand such as a lectin or an antibody.

As to antibodies, such specific reagents can be made using known techniques and using the target molecule as antigen. If the target molecule is poorly immunogenic, known methods for enhancing immunogenicity, such as, use of adjuvants, use of fragments of the target molecule as antigen, conjugating the target molecule or fragments thereof to a known carrier, such as albumin or keyhole limpet hemocyanin, immunizing immune cells in vitro and the like, as known in the art can be used. Either polyclonal or monoclonal antibodies can be made. For example, the 2G9 antibody reacts with the 15 kd TIA-1 protein which is related to apoptosis (Tian et al., 1991). Thus, by obtaining and using such antibodies, presence and amount of a target molecule can be determined using any of the known immunoassay formats.

The invention now will be described by reference to the following non-limiting examples.

EXAMPLE 1

A fusion protein consisting of the yeast GAL4 DNA-binding domain (amino acids 1-147) and E1B 19 kD protein (residues 2-175) expressed from a yeast shuttle vector (pMA424-19K or pAS-19K) was used as a bait in the two hybrid screen. The DNA sequences coding for the bait proteins were generated by PCR and cloned between the unique EcoRI and BamHI sites of pMA424 and pAS1 in the case of 19 kD (or between the EcoRI and SalI sites in the case of Bcl-2).

Plasmids expressing the 19 kD bait and a human cDNA expression library designated pACT (Durfee et al., 1993) tagged with the GAL4 activation domain (Durfee et al., 1993) were used for simultaneous transformation of yeast indicator strains, GGY1::171 (Gill and Ptashne, 1987) or Y153 (Durfee et al., 1993) by the lithium acetate method of Schiestl & Giest (1989). Functional reconstitution of the GAL4 transactivation function through interaction between the 19 kD moiety of the GAL4(1-147)-19K fusion protein and the cellular protein tagged with the activation domain of GAL4 activates expression of the lacZ reporter gene in strain GGY1::171 and both the HIS3 and lacZ genes in strain Y153.

Transfected cells were plated on synthetic dropout medium (SD), Rose et al., 1990, lacking leucine and histidine (strain GGY::171) or lacking histidine, tryptophan and leucine (strain Y153) by including 12.5 to 25 mM 3-AT 3-aminotriazole, a chemical which restores histidine auxotrophy (Kishore and Shaw, 1988). After three (GGY::171) to five (Y153) days at 30° C., colonies were lifted onto nitrocellulose filters (Schleicher and Schuell, BA85) and made permeable by freezing the filters for 5-10 sec on aluminum foil floats placed over liquid nitrogen.

Filters were overlaid on Whatman filters saturated with Z buffer (Miller, 1972) containing 1 mg/ml X-Gal and incubated at 30° C. (Breedan and Nasmyth, 1985). Colonies which turned blue within a period of 1-6 hr were picked and patched onto fresh selective plates.

The cells then were diluted in TE (10 mM Tris, pH 8.0 and 1 mM EDTA), plated onto selective plates and well-isolated colonies which were positive in the X-Gal assay were selected. Cells from the isolated colonies were patched onto selective plates and were then grown in selective liquid SD media. After overnight growth, the plasmid DNAs were extracted from the cells using glass beads (Hoffman and Winston, 1987) and the recovered DNA was used to transform an E. coli leuB strain (obtained from Stanley Fields, S.U.N.Y. Stony Brook) by electroporation using a BRL Cellporator according to the specifications of the manufacturer.

Ampicillin-resistant colonies were selected and replica plated on minimal M9 plates lacking amino acids to eliminate the plasmid expressing the bait protein. Plasmid DNA (containing the LEU marker) from colonies which grew on M9 media was prepared by alkaline lysis. The interaction between the 19 kD protein and the proteins coded by the cDNA clones was further ascertained by a second round of yeast transformation and X-gal staining. The cDNA clones which were positive with pMA-19K or pAS-19K and not with pMA424 or pAS1 vectors were selected for further analysis. The interaction of the selected cDNA clones with various heterologous protein baits then was determined to further ascertain the specificity of interaction with the 19 kD bait.

About 10⁵ transformants of each strain were screened for the activation of the reporter genes and three strongly positive clones were chosen for further analysis. The clones were tested in the two hybrid screen with either the 19K bait or with the GAL4 DNA binding domain vectors, pMA424 (Ma & Ptashne, 1987) or pAS1 (Durfee et al., 1993). All three clones (22, 44 and 91) reacted positively only with the 19K bait and not with the respective vectors.

The three clones (22, 44 and 91) were found to be defective in interaction with 19K mutant baits of interest.

EXAMPLE 2

To determine the specificity of the two hybrid interaction, the three clones, 22, 44 and 91, were tested against baits expressing several heterologous proteins (Table 1). Among the various heterologous protein baits used, the adenovirus E3 11.6 kD protein is of particular interest as that protein localizes on the nuclear envelope region and has an internal hydrophobic domain like the E1B 19 kD protein (Scaria et al., 1992). All three clones were negative against all the heterologous protein baits tested, indicating that the proteins coded by the clones specifically interact with the 19 kD protein.

                  TABLE 1     ______________________________________     Interaction of cellular apoptosis-regulating proteins     with heterologous protein baits.sup.@               lacZ expression (X-gal)     Bait        clone 22   clone 44   clone 91     ______________________________________     19 kD       B          B          B     HIV-1 Rev   W          W          W     HIV-1 Tat   W          W          W     HIV-1 Nef   w          w          W     HTLV-I Rex  W          W          W     SNF1        ND         W*         W*     E1A-Exon2   W          W          W     h-1amin     W          W          W     E3-11.6 kD  W          W          W     ______________________________________      B = Blue      W = White      .sup.@ Interactions with all baits except the yeast SNF1 protein were      carried out in yeast Strain GGY1::171. Interaction with SNF1 was carried      out in yeast strain Y153.      *Colonies which appeared on his, trp, leu plates after 6-8 days. Results      of Xgal staining are indicated as B, blue; W, white; or ND, not done.      The heterologous baits pLAM5 (human lamin) and pASSNF1 (yeast SNF1) have      been described (Bartel et al., 1993, Durfee et al., 1993). Baits pHIV1      Rev, pHIV1 Tat, pHIV1 Nef and pHTLV1 Rex expressing the various HIV or      HTLV regulating proteins were constructed as described herein and obtaine      from Cleta D'Sa Eipper and T. Subramanian, St. Louis University Medical      Center, St. Louis, MO.

EXAMPLE 3

Proteins coded by the various cDNA clones were prepared by in vitro transcription and translation using the phage T7-based expression vector, pET3b (Studier et al., 1990) and a commercially available kit (Promega, Madison, Wis.). ³⁵ S-labeled proteins coded by clones 22, 44 and 91 were incubated with protein extracts prepared from human 293 cells (Graham et al., 1977) which express abundant amounts of 19 kD protein or extracts of HeLa cells which do not express any adenovirus proteins. The interaction of the labeled proteins with the 19 kD protein then was analyzed by immunoprecipitation with an antibody specific for the 19 kD protein (Green et al., 1982). The 293 cell extract was used as a source of 19 kD protein rather than purified protein since the cell extract may be suitable to detect interaction of exogenously added proteins with 19 kD protein if the interaction occurs indirectly via other cellular proteins.

Exogenously added ³⁵ S-labeled proteins coded by both clones readily were precipitated with 19K antibodies in the presence of 293 cell extracts. Under the same binding conditions, ³⁵ S-labeled luciferase (control) was not precipitated. Similarly, no significant amounts of the various labeled proteins were precipitated from HeLa cell extracts.

In all cases, immunoprecipitations with normal rabbit serum also did not precipitate any detectable levels of the proteins.

The results indicate that the proteins coded by clones 22, 44 and 91, (hereafter referred to as 19K-interacting protein (Nip) 1, Nip2 and Nip3, respectively) specifically interact with the 19 kD protein either directly or indirectly and substantiate the results obtained by the two hybrid screens.

EXAMPLE 4

To confirm the interaction of the Nip proteins with the 19 kD protein in vivo, BSC40 were transfected with plasmid vectors expressing the 19 kD or various Nip proteins under the transcriptional control of the T7 promoter in a mammalian expression vector, pTM1 (Moss et al., 1990). The cells were infected with the recombinant vaccinia virus vTF7-3 expressing the T7 RNA polymerase gene (Fuerst et al., 1986) to induce the expression of proteins from the various pTM1 plasmids. To facilitate the analysis of protein interactions, the Nip proteins were tagged with an epitope corresponding to a 9 amino acid region of the haemagglutinin (HA) protein of influenza virus (Field et al., 1988). The pTM1 vector expressing only the HA epitope was used as a control.

Cells were labeled in vivo with a ³⁵ S-methionine and ³⁵ S-cysteine mixture, lysed and the lysates divided and subjected to immunoprecipitation analysis using either a monoclonal HA antibody 12CA5 (Boehringer-Mannheim, Indianapolis, Ind.) or a 19 kD antipeptide antibody (Green et al., 1982). The precipitated proteins were analyzed by SDS-PAGE and autoradiography to determine whether comparable levels of the 19 kD protein were present in each sample. The HA immunoprecipitates were separated by SDS-PAGE, blotted onto nitrocellulose and probed with the 19 kD antibody to detect the presence of coprecipitating 19 kD protein. A horseradish peroxidase-chemiluminescent detection system (ECL) (Amersham, Arlington Heights, Ill.) was used to visualize bound antibodies.

The 19 kD protein was not detected in HA immunoprecipitates from cells transfected with the pTM1-HA vector and pTM1-19 kD, but clearly was seen coprecipitating with HA-tagged Nip1, Nip2 and Nip3.

Cells also were cotransfected with plasmids expressing each of the Nip proteins and either the 19 kD or the vector plasmid. Cell lysates were immunoprecipitated with the 19 kD antibody, blotted and probed with anti-HA antibody. That experimentation yielded comparable results for Nip1 and Nip3 (Nip2 is not transfered to nitrocellulose under standard blotting conditions) and provides additional support for the specificity of the interactions.

The results indicate that the 19 kD protein interacts specifically with each of the Nip proteins in an in vivo immunoprecipitation assay.

EXAMPLE 5

To determine the subcellular localization of the 19 kD-interacting proteins and to investigate whether the proteins colocalize with the 19 kD protein within the cell, COS7 cells (Gluzman, 1981) with plasmids expressing the protein coding sequences of the cDNA clones either alone or with a 19 kD-expressing plasmid were overturned and the transfected cells were analyzed by indirect immunofluorescence. To facilitate the immunofluorescence analysis of the 19 kD-interacting proteins, the proteins first were tagged with an epitope corresponding to a 9 amino acid region of the hemagglutinin (HA) protein of influenza virus (Field et al., 1988).

The cDNA's were cloned into the expression plasmid pCMV-HA which tags the proteins with an HA epitope. COS7 cells, grown on 22 mm² coverslips in 30 mm dishes, were transfected with plasmids expressing the 19 kD protein (pRcCMV-19K) and/or each of the pCMV-HA based cDNA clones using the LipofectAMINE™ reagent (Gibco-BRL) following the manufacturer's protocols. Forty eight hours post-transfection cells were fixed with 3.7% formaldehyde in PBS and permeabilized with ice-cold methanol. Cells were double stained with mouse monoclonal anti-HA (Berkeley Antibody Co., Inc.) and rabbit polyclonal anti-19 kD peptide serum (Green et al., 1982), and visualized with goat anti-rabbit rhodamine conjugate (Cappel) and goat anti-mouse fluorescein conjugate (Pierce). Cells were observed and photographed for rhodamine (19 kD) and fluorescein (HA-tagged clones) fluorescence. The localization of each cellular protein was examined in the absence of or presence of the 19 kD protein. The 19 kD protein primarily was expressed at the nuclear envelope/endoplasmic reticulum region. Nip1 (clone 22) and Nip2 (clone 44) also appear to be concentrated primarily on the nuclear envelope region in addition to other cytoplasmic structures. Nip3 (clone 91) has a different pattern of localization having a punctate pattern resembling the patterns exhibited by mitochondria (Alberts et al., 1989).

In cells coexpressing Nip1, Nip2 or Nip3 and the 19 kD protein, both the 19 kD and each of the cellular proteins colocalized primarily at the nuclear envelope region. Hence, the apoptosis-regulating cellular proteins localize identically with the 19 kD protein, which includes a marked shift in the localization pattern of Nip3.

EXAMPLE 6

The two hybrid strategy provides a rapid method for genetic analysis of in vivo protein-protein interaction. A series of 19K mutants (Table 2) with regard to ability to protect against cell death induced by the DNA damaging agent cisplatin and to cooperate with E1A in transformation of primary cells were used (Subramanian et al., 1993). The wild type 19K sequence is identified as SEQ ID NO:8; the 14-5 mutant as SEQ ID NO:9; the 29-0 mutant as SEQ ID NO:10; the 50-1 mutant as SEQ ID NO:11; the 75-6 mutant as SEQ ID NO:12; the 90-6 mutant as SEQ ID NO:13; the 123-4 mutant as SEQ ID NO:14; and the 146-E mutant as SEQ ID NO:15.

In Table 2 the names of the various mutants are marked above the amino acid sequence and the amino acid changes are indicated below the sequence. The actual amino acids involved in the various mutations are indicated in boldface. Z indicates a stop codon. Δ indicates deletion. The underlined sequence is homologous to conserved domain I of bcl-2 (Williams & Smith, 1993).

                                      TABLE 2     __________________________________________________________________________     19K and Bcl-2 Mutants     __________________________________________________________________________     E1B 19K      ##STR1##      ##STR2##      ##STR3##     Bcl-2 (Seto et al., 1988)      ##STR4##      ##STR5##     121LTPFTARGCFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEY     181LNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK     __________________________________________________________________________

The 19K-coding regions of the mutants were cloned in the pMA424 vector and tested for ability to interact with Nip1, Nip2 and Nip3 in the two hybrid assay. The interaction was assessed by determining the level of lacZ expression by the X-gal filter blue/white color assay as well as by a quantitative ONPG-based β-galactosidase assay (Rose et al., 1990) (see Table 3).

                  TABLE 3     ______________________________________     Interaction of cellular apoptosis-regulating proteins     with 19K mutants     Cell        Interaction with cellular proteins     death       (Relative level of lacZ expression)     suppres-    Nip1       Nip2       Nip3     Mutant           sion.sup.@                     X-gal  ONPG  X-gal                                       ONPG  X-gal                                                  ONPG     ______________________________________     wt    +         B      1     B    1     B    1     14-5  (+)       B      0.61  B    0.55  LB   0.16     29-0  +         B      0.33  B    0.32  B    0.19     50-1  -         W      <0.01 W    0.09  W    0.01     75-6  +         B      1.72  B    1.60  B    0.22     90-6  -         W      <0.01 W    0.04  W    0.01     123-4 -         W      0.05  LB   0.24  LB   0.10     146-E +         B      0.87  B    0.98  B    0.28     ______________________________________      The protein interaction studies were carried out in yeast strain      GGY1::171. Results of Xgal staining are indicated as B, blue; W, white; o      LB, light blue.      .sup.@ Results on suppression of cell death are based on Subramanian et      al. (1993).      (+) Indicates positive at reduced levels.      β-galactosidase activities were determined from 3 to 4 independent      colonies selected at random and grown in liquid selective SD medium (Rose      et al., 1990). The β-galactosidase activity is expressed in units      described by Rose et al., 1990.

All three 19 kD-interacting proteins were negative for interaction with mutants 50-1 (amino acids 50-51) (SEQ ID NO:11) and 90-6 (amino acids 90-96) (SEQ ID NO:13). Nip1and Nip2 interacted at a reduced level with mutant 123-4 (substitutions for residues 123-124) (SEQ ID NO:14), but interacted more efficiently with the 19K mutant 75-6 (which carries a substitution for residues 75-76) (SEQ ID NO:12) than with wild-type (wt) 19K (SEQ ID NO:8).

Nip3 also exhibited a similar pattern of interaction; however, the overall level of expression of lacZ was lower. The studies identify two critical regions of the 19 kD protein marked by mutants 50-1 (SEQ ID NO:11) and 90-6 (SEQ ID NO:13) required for interaction with the cellular apoptosis-reducing proteins. In addition, sequences around residues 123-124 (mutant 123-4) (SEQ ID NO:14) also may influence the interactions.

The pattern of interaction of the three cellular proteins with the 19 kD protein is diagrammatically illustrated in FIG. 1. All the 19K mutants used make stable proteins in mammalian cells (Subramanian et al., 1993) and in yeast as GAL-4(1-147)-19 kD fusion proteins. Thus, the 19K mutants 50-1 (SEQ ID NO:11) and 90-6 (SEQ ID NO:13) defective for suppression of cell death, also are defective for interaction with the cellular proteins. The interaction of the cellular proteins with mutant 123-4 (SEQ ID NO:14) which also is defective in suppression of cell death is, in general, much reduced.

EXAMPLE 7

Since Bcl-2 functionally can substitute for 19K during adenovirus replication, the 19 kD-interacting cellular proteins also were shown to interact with the Bcl-2 protein.

The two hybrid analysis using a pMA424-based bait expressing the human Bcl-2 protein (Hockenbery et al., 1990) was used. Plasmids pMA-Bcl2 and pAS-Bcl2 express residues 1-239 of Bcl2. Yeast cells (GGY1::171) were transformed either with the pMA424-19K bait or with the pMA424-Bcl-2 bait along with each of the activation domain-tagged cDNA clones. The expression of lacZ was determined by the X-gal filter assay as well as by the liquid ONPG β-galactosidase assay (Table 4).

                  TABLE 4     ______________________________________     Interaction of 19 kD-interacting proteins with Bcl-2              Relative level of lacZ expression.sup.@              19 kD            Bcl-2     Protein    X-gal    ONPG      X-gal  ONPG     ______________________________________     Nip1       B        1         B      1.8     Nip2       B        1         B      0.7     Nip3       B        1         B      1.5     ______________________________________      .sup.@ Relative interaction is based on the β-galactosidase activity      expressed in cells expressing the respective tagged cDNA clones and the      19K bait.      B, blue.

All three clones interacted with the Bcl-2 bait. Nip1 and Nip3 in general interacted more efficiently with the Bcl-2 bait as compared with the 19 kD bait. The interaction of Nip2 was slightly lower with Bcl-2 than with 19 kD.

Proteins coded by the three cDNA clones were prepared by in vitro transcription and translation according to protocols supplied by commercial vendors and incubated with cell extracts prepared from CHO cells or CHO cells infected with an adenovirus recombinant that overexpresses the human Bcl-2 protein (Ad-Bcl2) (obtained and available from T. Subramanian and B. Tarodi, St. Louis University Medical Center, St. Louis, Mo.). The proteins were immunoprecipitated with a hamster monoclonal antibody specific for human Bcl-2 (Hockenbery et al., 1990) and analyzed by SDS-PAGE.

All three 19 kD-interacting proteins could be precipitated from extracts prepared from cells infected with the Ad-Bcl2 virus while in contrast there was no significant amount of proteins precipitated from uninfected cells.

To further substantiate the interaction of the Nip proteins with Bcl-2, BSC40 cells infected with vTF7-3 (Fuerst et al., 1986) expressing the T7 RNA polymerase gene and cotransfected with plasmids expressing Bcl-2 and one of the HA-tagged Nip proteins were labeled, lysed and analyzed essentially as described in Example 4 for the 19 kD protein. Comparable levels of Bcl-2 protein were present in each sample as determined by immunoprecipitation using the Bcl-2 monoclonal antibody 6C8 (Hockenbery et al., 1990). Each sample was subjected to immunoprecipitation using the HA monoclonal antibody 12CA5 (Boehringer-Mannheim); the precipitated proteins were separated by SDS-PAGE, blotted and probed for the presence of Bcl-2. Bcl-2 clearly was coprecipitated with HA-Nip1, HA-Nip2 and HA-Nip3.

An alternate experiment in which the cells were cotransfected with each of the Nip proteins and either Bcl-2 or the vector plasmid and cell lysates were immunoprecipitated with antibody directed against the Bcl-2 protein and probed with anti-HA antibody, yielded comparable results for Nip1 and Nip3 (Nip2 is not transfered to nitrocellulose under standard blotting conditions) and provides additional support for the specificity of the interactions. Thus, using in vivo coimmunoprecipitation, a specific interaction between each of the Nip proteins and Bcl-2 was found. The results, together with the in vitro immunoprecipitation data, indicate that the three Nip proteins interact with Bcl-2 as well as with the 19 kD proteins.

Using bcl-2 as the bait in the two hybrid analysis, three additional regulating proteins (Bip for bcl-2 interacting protein) were identified. Two of the clones (Bip 1A, SEQ ID NOS:26-27 and Bip 13, SEQ ID NOS:30-31) also interact with the 19K bait, while clone Bip 5, SEQ ID NOS:28-29, did not. Bip 1A was tested for interaction with the 19K mutants as described in Example 5 and exhibited a similar pattern of interaction to that seen with NIP's 1, 2 and 3. Interaction was negative with mutant 90-6 (SEQ ID NO:13) and greatly diminished with mutant 50-1 (SEQ ID NO:11), as illustrated in FIG. 1.

EXAMPLE 8

The observation that the various 19 kD-interacting proteins also interact with the Bcl-2 protein is significant since the primary amino acid sequence of the 19 kD protein is not significantly homologous to Bcl-2 and related proteins (reviewed by Williams and Smith, 1993). Since four cellular proteins tested (Nip1, Nip2, Nip3 and Bip1A) failed to interact with 19K mutants 50-1 (SEQ ID NO:11) and 90-6 (SEQ ID NO:13), regions of homology corresponding to the mutated regions of 19 kD protein must exist. Three regions of homology between the amino acid sequences encompassing the 19K mutants (50-1 and 90-6) and Bcl-2 were identified.

Based thereon, three different deletion mutants within the Bcl-2 (SEQ ID NO:16) coding region where constructed by oligonucleotide-directed mutagenesis using commerically available kits (Amersham) (Table 2). The mutants lack Bcl-2 (SEQ ID NO:16) residues 42 to 48 (#42-8) (SEQ ID NO:17), 80 to 86 (#80-6) (SEQ ID NO:18) and 106 to 115 (#106-5) (SEQ ID NO:19) of Bcl-2. The mutants were cloned in pMA424 and used in the two hybrid assay. Interaction was determined by the lacZ expression assays (Table 5).

                  TABLE 5     ______________________________________     Interaction of 19 kD-interacting proteins with Bcl-2 mutants            Relative level of lacZ expression.sup.@            Nip1      Nip2        Nip3     Mutant   X-gal   ONPG    X-gal ONPG  X-gal ONPG     ______________________________________     wt       B       1.0     B     1.0   B     1.0     42-8     W       0.16    W     0.09  W     0.19     80-6     B       1.21    B     0.80  B     2.87     106-5    w       0.01    W     0.06  W     0.11     ______________________________________      .sup.@ Relative interaction is based on the β-galactosidase activity      expressed in cells expressing the respective tagged cDNA clones and the      Bcl2 baits expressing the various mutants.      B, blue; W, white.

Mutants 42-8 and 106-5 were defective for interaction with the three clones tested while mutant 80-6 was not. Since the amino acid sequences of Bcl-2 deleted in mutants 42-8 and 106-5 appear to be homologous to sequences around 19K mutants 90-6 and 50-1, respectively, the 19 kD-interacting proteins recognize bipartite sequence motifs common to both the 19 kD and Bcl-2 proteins (FIG. 1).

EXAMPLE 9

The DNA sequences of the six cDNA clones were determined by dideoxy sequencing using the Sequenase kit (U.S. Biochemicals) of pAct-based plasmids (Durfee et al., 1993) or after subcloning into the pBluescript II KS(+) vector (Stratagene, San Diego, Calif.) using Sequenase version 2 (U.S. Biochemical Corp.). Sequencing first was initiated with primers complementary to sequences upstream of the cDNA cloning site in pAct or the T3 promoter of pBluescript. Subsequently, other primers were synthesized on the basis of newly determined sequences and used for sequencing both strands. The reading frame in relation to the GAL4 activation domain was established.

Since the activation domain is tagged at the 5' end of the cDNA's, a fraction of cDNA's isolated by the two hybrid system may be lacking the sequences corresponding to native 5' ends of the various mRNA's. Hence, the 5'-upstream sequences corresponding to the three isolated cDNA clones were obtained by reverse transcription of poly(A)-containing RNA prepared from human HeLa cells or from human placenta and PCR amplification (Frohman et al., 1988) using two nested primers (5'RACE) using a commercially available kit (5'AmpliFINDER, Clonetech) or from cDNA clones isolated from a human B cell cDNA library (Ausubel et al., 1992).

Downstream sequences were obtained by 3'RACE (described hereinbelow) using a commercially available kit (GibcoBRL). The Bluescript-based cDNA clones were prepared after infection with the helper phage and sequenced using appropriate primers. The DNA fragments obtained by RACE were cloned and sequenced.

The RACE analysis provided additional DNA sequences corresponding to three codons including an ATG codon for clone 22 (Nip1) while extensive analysis did not provide additional protein coding sequences corresponding to cDNA clones 44 (Nip2) and 91 (Nip3). The coding region of clone 44 at the 3' end (30 amino acids) was obtained by 3' RACE analysis.

For Bip5, 139 bp of 5' sequences were obtained as follows. The two hybrid clone contained nucleotides 109 to 1542. Comparison of the Bip5 sequence with a human fetal brain cDNA clone revealed substantial sequence identity and overlap (clone T08302, EST 06193, Adams et al., 1993) between the two clones. Clone T08302 contains a 366 bp fragment and includes an open reading frame beginning at the ATG of the clone. T08302 was ligated to the 5' end of the Bip5 clone following PCR amplification.

EXAMPLE 10

The amino acid sequences of Nip1 (SEQ ID NO:21), Nip2 (SEQ ID NO:23), Nip3 (SEQ ID NO:25), Bip1A (SEQ ID NO:27), Bip5 (SEQ ID NO:29) and Bip13 (SEQ ID NO:31) based on DNA sequences are presented in Table 6. Putative transmembrane domains of Nip1and Nip3 are underlined.

The amino acid sequences were analyzed to determine if any similarity to other sequences existed in various known and available data banks (GenBank, PIR and SwissProt National Center for Biotechnology Information (NCBI) using the FASTA, TFASTA programs (Pearson & Lipman 1988) available in the University of Wisconsin Genetic Computer group (UWGCG package) and BLAST (NCBI) (Alschul et al., 1990) algorithms. The sequences also were analyzed by the PROSITE (UWGCG Software package) program to identify functional sequence motifs.

Nip1(SEQ ID NO:21) is a 228 amino acid protein and contains a putative membrane-spanning hydrophobic domain. The presence of the membrane-spanning domain enables the protein to form stable associations with cellular membranes and accounts for the observed localization of the protein to the nuclear envelope/endoplasmic reticulum region of the cell.

Sequence comparison of Nip1(SEQ ID NO:21) indicated that the protein is not significantly homologous to other known proteins. However, a 59 to 83 amino acid region of Nip1 has homology (29 to 36 per cent identity and 55 to 60 per cent similarity) to a conserved region (FIG. 2A) within the catalytic domain of three mammalian (rat, mouse and cow) calmodulin-dependent 3'-5'-cyclic nucleotide phosphodiesterases (PDE) (SEQ ID NO:34) (Bentley et al., 1992; Polli et al., 1992; Repaske et al., 1992).

Because Nip1 is likely to possess an enzymatic activity, Nip1 can be used as a diagnostic tool to ascertain the state of the cell. Hence, excess of reduced Nip1 phosphodiesterase activity can be a means for monitoring the metabolic state of a cell, tissue or organ.

Nip2 (SEQ ID NO:23) is a 314 amino acid protein. Sequence comparison revealed that a 126 amino acid region of Nip2 shares significant homology (47 per cent identity and 66 per cent similarity; FIG. 2C) to the human GTPase activating protein RhoGAP (SEQ ID NO:35) (Barfod et al., 1993; Lancaster et al., 1994). The homology between Nip2 and RhoGAP does not extend to the functional domain of RhoGAP and is located in an upstream region which has been postulated to be removed by proteolysis (Barfod et al., 1993).

A scrutiny of the Nip2 sequence also revealed the presence of a putative Ca⁺⁺ --binding motif (amino acids 79-90 of SEQ ID NO:23) (FIG. 2B).

Even though Nip2 does not contain a membrane-spanning hydrophobic domain, indirect immunofluorescence analysis indicates that it is associated with cytoplasmic structures.

Nip3 (SEQ ID NO:25) is a 194 amino acid protein and contains a presumptive membrane-spanning hydrophobic domain. Nip3 also appears to be a novel protein. However, the cDNA sequence is highly homologous to a rat cDNA clone which encodes the rat brain calbindin-D protein (Hunziker and Schrickel, 1988). A smaller cDNA clone isolated from human fetal brain (Adams et al., 1992) is identical to sequences within the Nip3 coding region. cDNA clone 91 (Nip3) and the fetal brain cDNA do not contain the coding sequences for calbindin. Similarly, two other cDNA clones containing Nip3 sequences isolated from a human B cell cDNA library do not contain the calbindin coding sequences. An extensive search for additional sequences in DNA amplified by 5'RACE of human placental RNA with primers specific for clone 91 did not yield additional 5' sequences corresponding to calbindin coding sequences.

Thus, Nip3 is coded by a unique mRNA in human cells. That notion is in agreement with the observation that rat calbindin cDNA hybridizes to multiple mRNA species from human membrane (Iacopino and Christakos, 1990).

All three cellular proteins contain sequence motifs designated PEST (enriched in proline, glutamic acid, serine and threonine) (SEQ ID NO:33) sequences (Rogers et al, 1986) suggesting that the proteins may be degraded rapidly and expressed in a stage-specific manner (Rogers et al., 1986).

Bip1A (SEQ ID NO:27) is a 160 amino acid protein.

Bip5 (SEQ ID NO:29) is a 259 amino acid protein and does not exhibit significant homology to other known proteins.

On sequencing, it was determined that clone Bip13 (SEQ ID NO:31) bears a high degree of homology with CD22 (Wilson et al., 1991; Stamenkovic and Seed, 1990). CD22 is known to increase intracellular calcium and may be a B cell activator, however, the role of CD22 in apoptosis heretofore was unknown.

                                      TABLE 6     __________________________________________________________________________     Amino Acid and Nucliec Acid Sequences     __________________________________________________________________________     A. Nip1      ##STR6##      ##STR7##      ##STR8##      ##STR9##      ##STR10##      ##STR11##      ##STR12##      ##STR13##      ##STR14##      ##STR15##      ##STR16##      ##STR17##      ##STR18##     B. Nip2      ##STR19##      ##STR20##      ##STR21##      ##STR22##      ##STR23##      ##STR24##      ##STR25##      ##STR26##      ##STR27##      ##STR28##      ##STR29##      ##STR30##      ##STR31##      ##STR32##      ##STR33##      ##STR34##      ##STR35##      ##STR36##     C. Nip3      ##STR37##      ##STR38##      ##STR39##      ##STR40##      ##STR41##      ##STR42##      ##STR43##      ##STR44##      ##STR45##      ##STR46##      ##STR47##      ##STR48##     D. Bip1A      ##STR49##      ##STR50##      ##STR51##      ##STR52##      ##STR53##      ##STR54##      ##STR55##      ##STR56##      ##STR57##      ##STR58##     E. Bip5      ##STR59##      ##STR60##      ##STR61##      ##STR62##      ##STR63##      ##STR64##      ##STR65##      ##STR66##      ##STR67##      ##STR68##      ##STR69##      ##STR70##      ##STR71##      ##STR72##      ##STR73##      ##STR74##      ##STR75##      ##STR76##      ##STR77##     F. Bip13      ##STR78##      ##STR79##      ##STR80##      ##STR81##      ##STR82##      ##STR83##      ##STR84##      ##STR85##      ##STR86##      ##STR87##      ##STR88##      ##STR89##      ##STR90##      ##STR91##      ##STR92##      ##STR93##      ##STR94##      ##STR95##      ##STR96##      ##STR97##      ##STR98##      ##STR99##      ##STR100##      ##STR101##      ##STR102##      ##STR103##      ##STR104##     __________________________________________________________________________

EXAMPLE 11

The cellular apoptosis-regulating proteins also interact with the BHRF-1 protein of Epstein-Barr virus. BHRF-1 (Baer et al., 1984; Pearson et al., 1987) shares significant homology with the Bcl-2 protein (Cleary et al., 1986; Williams and Smith, 1993). The BHRF-1 protein can protect against cell death induced by certain apoptosis-inducing stimuli such as serum depletion and the DNA damaging agents (Henderson et al., 1993). Sequences homologous to the 19 kD motifs are present in the BHRF-1 protein. The two domains of the 19 kD and Bcl-2 proteins required for interaction with the cellular proteins are absent in Bax, a homolog of Bcl-2 (Oltvai et al., 1993). The absence of the two domains in Bax, which has a cell death promoting activity, supports the observation that the two domains are important for mediating cell survival by the 19 kD and Bcl-2 proteins.

The 19 kD protein of Ad2 and Ad5 contains a region (residues 60-69) (see Table 1) homologous to a conserved domain (termed domain I) present in various Bcl-2-related proteins, including Bax (Williams and Smith, 1993; Oltvai et al., 1993). Nip1, Nip2 and Nip3 do not interact with human Bax, which contains domain I, in the yeast two hybrid assay suggesting that the conserved domain I may not be essential for interaction of Nip1, Nip2 and Nip3. However, Bcl-2 can suppress cell death promoted by Bax, possibly by forming Bax-Bcl-2 heterodimers (Oltvai et al., 1993).

In the nematode C. elegans, apoptotic cell death is inhibited by a survival promoting gene designated ced-9 (Hengartner et al., 1992). The ced-9 gene antagonizes the activity of two nematode cell death-inducing genes, ced-3 and ced-4, which are activated during programmed cell death (Yuan and Horvitz, 1990). Bcl-2 can substitute functionally for ced-9 in C. elegans (Vaux et al., 1991).

The mammalian IL-1β-converting enzyme (ICE), a homolog of ced-3 (Yuan et al., 1993; Miura et al., 1993), induces cell death in mammalian cells. The activity of ICE can be suppressed by Bcl-2 (Miura et al., 1993) although it is not known whether there is any physical interaction between ICE and Bcl-2.

EXAMPLE 12

The homology between Nip2 and Rho GTPase-activating protein (RhoGAP) raises the possibility that the signal transduction pathway may play a key role in counteracting the cell death inducing stimuli. In this context, it has recently been reported that the Bcl-2 protein associates with R-ras (Fernandez-Sarabia & Bischoff, 1993), a member of Ras superfamily. Nip2 may modulate the activity of RhoGAP through alternate mechanisms such a formation of RhoGAP-Nip2 heterodimers. Because the homology between Nip2 and RhoGAP is located upstream of the N-terminus of active RhoGAP (Garrett et al., 1991), Nip2 might affect the activity of RhoGAP by interfering with the processing of RhoGAP.

Nip2 has a putative Ca⁺² --binding motif. Since intracellular Ca⁺² appears to be an important mediator of apoptotic cell death, it is possible that the activity of Nip2 may be modulated by Ca⁺². Hence, interactions between the 19 Kd protein and Nip2 could remove the influence thereof on RhoGAP activity, thus affecting p21^(rho) and potentially other members of the ras superfamily.

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Yuan, J. et al., (1993), Cell 75, 641-652

All references cited herein are incorporated herein by reference in entirety.

As will be evident to the artisan, various modifications and changes can be made without departing from the spirit and scope of the instant invention.

    __________________________________________________________________________     SEQUENCE LISTING     (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 35     (2) INFORMATION FOR SEQ ID NO:1:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     AlaAlaProAlaProGlyPhePheSer     15     (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     TyrArgArgAspPheAlaGlu     15     (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     TyrLysTrpGluPheGluGlu     15     (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     AlaAlaAlaAlaValAlaPheLeuSer     15     (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (ix) FEATURE:     (A) NAME/KEY: Modified-site     (B) LOCATION: 2     (ix) FEATURE:     (A) NAME/KEY: Modified-site     (B) LOCATION: 3     (ix) FEATURE:     (A) NAME/KEY: Modified-site     (B) LOCATION: 4     (ix) FEATURE:     (A) NAME/KEY: Modified-site     (B) LOCATION: 6     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     TyrXaaXaaXaaPheXaaGlu     15     (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 8 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (ix) FEATURE:     (A) NAME/KEY: Modified-site     (B) LOCATION: 3     (ix) FEATURE:     (A) NAME/KEY: Modified-site     (B) LOCATION: 5     (ix) FEATURE:     (A) NAME/KEY: Modified-site     (B) LOCATION: 7     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     AlaAlaXaaAlaXaaPheXaaSer     15     (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     AlaAlaProAlaProGlyIlePheSer     15     (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 175 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     MetGluAlaTrpGluCysLeuGluAspPheSerAlaValArgAsnLeu     151015     LeuGluGlnSerSerAsnSerThrSerTrpPheTrpArgPheLeuTrp     202530     GlySerSerGlnAlaLysLeuValCysArgIleLysGluAspTyrLys     354045     TrpGluPheGluGluLeuLeuLysSerCysGlyGluLeuPheAspSer     505560     LeuAsnLeuGlyHisGlnAlaLeuPheGlnGluLysValIleLysThr     65707580     LeuAspPheSerThrProGlyArgAlaAlaAlaAlaValAlaPheLeu     859095     SerPheIleLysAspLysTrpSerGluGluThrHisLeuSerGlyGly     100105110     TyrLeuLeuAspPheLeuAlaMetHisLeuTrpArgAlaValValArg     115120125     HisLysAsnArgLeuLeuLeuLeuSerSerValArgProAlaIleIle     130135140     ProThrGluGluGlnGlnGlnGluGluAlaArgArgArgArgArgGln     145150155160     GluGlnSerProTrpAsnProArgAlaGlyLeuAspProArgGlu     165170175     (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 175 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     MetGluAlaTrpGluCysLeuGluAspPheSerAlaValAlaSerLeu     151015     LeuGluGlnSerSerAsnSerThrSerTrpPheTrpArgPheLeuTrp     202530     GlySerSerGlnAlaLysLeuValCysArgIleLysGluAspTyrLys     354045     TrpGluPheGluGluLeuLeuLysSerCysGlyGluLeuPheAspSer     505560     LeuAsnLeuGlyHisGlnAlaLeuPheGlnGluLysValIleLysThr     65707580     LeuAspPheSerThrProGlyArgAlaAlaAlaAlaValAlaPheLeu     859095     SerPheIleLysAspLysTrpSerGluGluThrHisLeuSerGlyGly     100105110     TyrLeuLeuAspPheLeuAlaMetHisLeuTrpArgAlaValValArg     115120125     HisLysAsnArgLeuLeuLeuLeuSerSerValArgProAlaIleIle     130135140     ProThrGluGluGlnGlnGlnGluGluAlaArgArgArgArgArgGln     145150155160     GluGlnSerProTrpAsnProArgAlaGlyLeuAspProArgGlu     165170175     (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 175 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     MetGluAlaTrpGluCysLeuGluAspPheSerAlaValArgAsnLeu     151015     LeuGluGlnSerSerAsnSerThrSerTrpPheTrpAlaSerLeuTrp     202530     GlySerSerGlnAlaLysLeuValCysArgIleLysGluAspTyrLys     354045     TrpGluPheGluGluLeuLeuLysSerCysGlyGluLeuPheAspSer     505560     LeuAsnLeuGlyHisGlnAlaLeuPheGlnGluLysValIleLysThr     65707580     LeuAspPheSerThrProGlyArgAlaAlaAlaAlaValAlaPheLeu     859095     SerPheIleLysAspLysTrpSerGluGluThrHisLeuSerGlyGly     100105110     TyrLeuLeuAspPheLeuAlaMetHisLeuTrpArgAlaValValArg     115120125     HisLysAsnArgLeuLeuLeuLeuSerSerValArgProAlaIleIle     130135140     ProThrGluGluGlnGlnGlnGluGluAlaArgArgArgArgArgGln     145150155160     GluGlnSerProTrpAsnProArgAlaGlyLeuAspProArgGlu     165170175     (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 175 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     MetGluAlaTrpGluCysLeuGluAspPheSerAlaValArgAsnLeu     151015     LeuGluGlnSerSerAsnSerThrSerTrpPheTrpArgPheLeuTrp     202530     GlySerSerGlnAlaLysLeuValCysArgIleLysGluAspTyrLys     354045     TrpAlaSerGluGluLeuLeuLysSerCysGlyGluLeuPheAspSer     505560     LeuAsnLeuGlyHisGlnAlaLeuPheGlnGluLysValIleLysThr     65707580     LeuAspPheSerThrProGlyArgAlaAlaAlaAlaValAlaPheLeu     859095     SerPheIleLysAspLysTrpSerGluGluThrHisLeuSerGlyGly     100105110     TyrLeuLeuAspPheLeuAlaMetHisLeuTrpArgAlaValValArg     115120125     HisLysAsnArgLeuLeuLeuLeuSerSerValArgProAlaIleIle     130135140     ProThrGluGluGlnGlnGlnGluGluAlaArgArgArgArgArgGln     145150155160     GluGlnSerProTrpAsnProArgAlaGlyLeuAspProArgGlu     165170175     (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 175 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     MetGluAlaTrpGluCysLeuGluAspPheSerAlaValArgAsnLeu     151015     LeuGluGlnSerSerAsnSerThrSerTrpPheTrpArgPheLeuTrp     202530     GlySerSerGlnAlaLysLeuValCysArgIleLysGluAspTyrLys     354045     TrpGluPheGluGluLeuLeuLysSerCysGlyGluLeuPheAspSer     505560     LeuAsnLeuGlyHisGlnAlaLeuPheGlnAlaSerValIleLysThr     65707580     LeuAspPheSerThrProGlyArgAlaAlaAlaAlaValAlaPheLeu     859095     SerPheIleLysAspLysTrpSerGluGluThrHisLeuSerGlyGly     100105110     TyrLeuLeuAspPheLeuAlaMetHisLeuTrpArgAlaValValArg     115120125     HisLysAsnArgLeuLeuLeuLeuSerSerValArgProAlaIleIle     130135140     ProThrGluGluGlnGlnGlnGluGluAlaArgArgArgArgArgGln     145150155160     GluGlnSerProTrpAsnProArgAlaGlyLeuAspProArgGlu     165170175     (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 168 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     MetGluAlaTrpGluCysLeuGluAspPheSerAlaValArgAsnLeu     151015     LeuGluGlnSerSerAsnSerThrSerTrpPheTrpArgPheLeuTrp     202530     GlySerSerGlnAlaLysLeuValCysArgIleLysGluAspTyrLys     354045     TrpGluPheGluGluLeuLeuLysSerCysGlyGluLeuPheAspSer     505560     LeuAsnLeuGlyHisGlnAlaLeuPheGlnGluLysValIleLysThr     65707580     LeuAspPheSerThrProGlyArgAlaSerPheIleLysAspLysTrp     859095     SerGluGluThrHisLeuSerGlyGlyTyrLeuLeuAspPheLeuAla     100105110     MetHisLeuTrpArgAlaValValArgHisLysAsnArgLeuLeuLeu     115120125     LeuSerSerValArgProAlaIleIleProThrGluGluGlnGlnGln     130135140     GluGluAlaArgArgArgArgArgGlnGluGlnSerProTrpAsnPro     145150155160     ArgAlaGlyLeuAspProArgGlu     165     (2) INFORMATION FOR SEQ ID NO:14:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 175 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     MetGluAlaTrpGluCysLeuGluAspPheSerAlaValArgAsnLeu     151015     LeuGluGlnSerSerAsnSerThrSerTrpPheTrpArgPheLeuTrp     202530     GlySerSerGlnAlaLysLeuValCysArgIleLysGluAspTyrLys     354045     TrpGluPheGluGluLeuLeuLysSerCysGlyGluLeuPheAspSer     505560     LeuAsnLeuGlyHisGlnAlaLeuPheGlnGluLysValIleLysThr     65707580     LeuAspPheSerThrProGlyArgAlaAlaAlaAlaValAlaPheLeu     859095     SerPheIleLysAspLysTrpSerGluGluThrHisLeuSerGlyGly     100105110     TyrLeuLeuAspPheLeuAlaMetHisLeuAlaSerAlaValValArg     115120125     HisLysAsnArgLeuLeuLeuLeuSerSerValArgProAlaIleIle     130135140     ProThrGluGluGlnGlnGlnGluGluAlaArgArgArgArgArgGln     145150155160     GluGlnSerProTrpAsnProArgAlaGlyLeuAspProArgGlu     165170175     (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 147 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     MetGluAlaTrpGluCysLeuGluAspPheSerAlaValArgAsnLeu     151015     LeuGluGlnSerSerAsnSerThrSerTrpPheTrpArgPheLeuTrp     202530     GlySerSerGlnAlaLysLeuValCysArgIleLysGluAspTyrLys     354045     TrpGluPheGluGluLeuLeuLysSerCysGlyGluLeuPheAspSer     505560     LeuAsnLeuGlyHisGlnAlaLeuPheGlnGluLysValIleLysThr     65707580     LeuAspPheSerThrProGlyArgAlaAlaAlaAlaValAlaPheLeu     859095     SerPheIleLysAspLysTrpSerGluGluThrHisLeuSerGlyGly     100105110     TyrLeuLeuAspPheLeuAlaMetHisLeuTrpArgAlaValValArg     115120125     HisLysAsnArgLeuLeuLeuLeuSerSerValArgProAlaIleIle     130135140     ProThrSer     145     (2) INFORMATION FOR SEQ ID NO:16:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 239 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     MetAlaHisAlaGlyArgSerGlyTyrAspAsnArgGluIleValMet     151015     LysTyrIleHisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAla     202530     GlyAspValGlyAlaAlaProProGlyAlaAlaProAlaProGlyPhe     354045     PheSerSerGlnProGlyHisThrProHisProAlaAlaSerArgAsp     505560     ProValAlaArgThrSerProLeuGlnThrProAlaAlaProGlyAla     65707580     AlaAlaGlyProAlaLeuSerProValProProValValHisLeuThr     859095     LeuArgGlnAlaGlyAspAspPheSerArgArgTyrArgArgAspPhe     100105110     AlaGluMetSerSerGlnLeuHisLeuThrProPheThrAlaArgGly     115120125     CysPheAlaThrValValGluGluLeuPheArgAspGlyValAsnTrp     130135140     GlyArgIleValAlaPhePheGluPheGlyGlyValMetCysValGlu     145150155160     SerValAsnArgGluMetSerProLeuValAspAsnIleAlaLeuTrp     165170175     MetThrGluTyrLeuAsnArgHisLeuHisThrTrpIleGlnAspAsn     180185190     GlyGlyTrpAspAlaPheValGluLeuTyrGlyProSerMetArgPro     195200205     LeuPheAspPheSerTrpLeuSerLeuLysThrLeuLeuSerLeuAla     210215220     LeuValGlyAlaCysIleThrLeuGlyAlaTyrLeuGlyHisLys     225230235     (2) INFORMATION FOR SEQ ID NO:17:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 232 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     MetAlaHisAlaGlyArgSerGlyTyrAspAsnArgGluIleValMet     151015     LysTyrIleHisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAla     202530     GlyAspValGlyAlaAlaProProGlyPheSerSerGlnProGlyHis     354045     ThrProHisProAlaAlaSerArgAspProValAlaArgThrSerPro     505560     LeuGlnThrProAlaAlaProGlyAlaAlaAlaGlyProAlaLeuSer     65707580     ProValProProValValHisLeuThrLeuArgGlnAlaGlyAspAsp     859095     PheSerArgArgTyrArgArgAspPheAlaGluMetSerSerGlnLeu     100105110     HisLeuThrProPheThrAlaArgGlyCysPheAlaThrValValGlu     115120125     GluLeuPheArgAspGlyValAsnTrpGlyArgIleValAlaPhePhe     130135140     GluPheGlyGlyValMetCysValGluSerValAsnArgGluMetSer     145150155160     ProLeuValAspAsnIleAlaLeuTrpMetThrGluTyrLeuAsnArg     165170175     HisLeuHisThrTrpIleGlnAspAsnGlyGlyTrpAspAlaPheVal     180185190     GluLeuTyrGlyProSerMetArgProLeuPheAspPheSerTrpLeu     195200205     SerLeuLysThrLeuLeuSerLeuAlaLeuValGlyAlaCysIleThr     210215220     LeuGlyAlaTyrLeuGlyHisLys     225230     (2) INFORMATION FOR SEQ ID NO:18:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 232 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:     MetAlaHisAlaGlyArgSerGlyTyrAspAsnArgGluIleValMet     151015     LysTyrIleHisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAla     202530     GlyAspValGlyAlaAlaProProGlyAlaAlaProAlaProGlyPhe     354045     PheSerSerGlnProGlyHisThrProHisProAlaAlaSerArgAsp     505560     ProValAlaArgThrSerProLeuGlnThrProAlaAlaProGlySer     65707580     ProValProProValValHisLeuThrLeuArgGlnAlaGlyAspAsp     859095     PheSerArgArgTyrArgArgAspPheAlaGluMetSerSerGlnLeu     100105110     HisLeuThrProPheThrAlaArgGlyCysPheAlaThrValValGlu     115120125     GluLeuPheArgAspGlyValAsnTrpGlyArgIleValAlaPhePhe     130135140     GluPheGlyGlyValMetCysValGluSerValAsnArgGluMetSer     145150155160     ProLeuValAspAsnIleAlaLeuTrpMetThrGluTyrLeuAsnArg     165170175     HisLeuHisThrTrpIleGlnAspAsnGlyGlyTrpAspAlaPheVal     180185190     GluLeuTyrGlyProSerMetArgProLeuPheAspPheSerTrpLeu     195200205     SerLeuLysThrLeuLeuSerLeuAlaLeuValGlyAlaCysIleThr     210215220     LeuGlyAlaTyrLeuGlyHisLys     225230     (2) INFORMATION FOR SEQ ID NO:19:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 229 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:     MetAlaHisAlaGlyArgSerGlyTyrAspAsnArgGluIleValMet     151015     LysTyrIleHisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAla     202530     GlyAspValGlyAlaAlaProProGlyAlaAlaProAlaProGlyPhe     354045     PheSerSerGlnProGlyHisThrProHisProAlaAlaSerArgAsp     505560     ProValAlaArgThrSerProLeuGlnThrProAlaAlaProGlyAla     65707580     AlaAlaGlyProAlaLeuSerProValProProValValHisLeuThr     859095     LeuArgGlnAlaGlyAspAspPheSerSerSerGlnLeuHisLeuThr     100105110     ProPheThrAlaArgGlyCysPheAlaThrValValGluGluLeuPhe     115120125     ArgAspGlyValAsnTrpGlyArgIleValAlaPhePheGluPheGly     130135140     GlyValMetCysValGluSerValAsnArgGluMetSerProLeuVal     145150155160     AspAsnIleAlaLeuTrpMetThrGluTyrLeuAsnArgHisLeuHis     165170175     ThrTrpIleGlnAspAsnGlyGlyTrpAspAlaPheValGluLeuTyr     180185190     GlyProSerMetArgProLeuPheAspPheSerTrpLeuSerLeuLys     195200205     ThrLeuLeuSerLeuAlaLeuValGlyAlaCysIleThrLeuGlyAla     210215220     TyrLeuGlyHisLys     225     (2) INFORMATION FOR SEQ ID NO:20:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1101 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 11..694     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:     AGTCCCCAACATGGCGGCTCCCCAAGACGTCCACGTCCGGATCTGTAAC49     MetAlaAlaProGlnAspValHisValArgIleCysAsn     1510     CAAGAGATTGTCAAATTTGACCTGGAGGTGAAGGCGCTTATTCAGGAT97     GlnGluIleValLysPheAspLeuGluValLysAlaLeuIleGlnAsp     152025     ATCCGTGATTGTTCAGGACCCTTAAGTGCTCTTACTGAACTGAATACT145     IleArgAspCysSerGlyProLeuSerAlaLeuThrGluLeuAsnThr     30354045     AAAGTAAAAGAGAAATTTCAACAGTTGCGTCACAGAATACAGGACCTG193     LysValLysGluLysPheGlnGlnLeuArgHisArgIleGlnAspLeu     505560     GAGCAGTTGGCTAAAGAGCAAGACAAAGAATCAGAGAAACAACTTCTA241     GluGlnLeuAlaLysGluGlnAspLysGluSerGluLysGlnLeuLeu     657075     CTCCAGGAAGTGGAGAATCACAAAAAGCAGATGCTCAGCAATCAGGCC289     LeuGlnGluValGluAsnHisLysLysGlnMetLeuSerAsnGlnAla     808590     TCATGGAGGAAAGCTAATCTCACCTGCAAAATTGCAATCGACAATCTA337     SerTrpArgLysAlaAsnLeuThrCysLysIleAlaIleAspAsnLeu     95100105     GAGAAAGCAGAACTTCTTCAGGGAGGAGATCTCTTAAGGCAAAGGAAA385     GluLysAlaGluLeuLeuGlnGlyGlyAspLeuLeuArgGlnArgLys     110115120125     ACCACCAAAGAGAGCCTGGCCCAGACATCCAGTACCATCACTGAGAGC433     ThrThrLysGluSerLeuAlaGlnThrSerSerThrIleThrGluSer     130135140     CTCATGGGGATCAGCAGGATGATGGCCCAGCAGGTCCAGCAGAGCGAG481     LeuMetGlyIleSerArgMetMetAlaGlnGlnValGlnGlnSerGlu     145150155     GAGGCCATGCAGTCTCTAGTCACTTCTTCACGAACGATCCTGGATGCA529     GluAlaMetGlnSerLeuValThrSerSerArgThrIleLeuAspAla     160165170     AATGAAGAATTTAAGTCCATGTCGGGCACCATCCAGCTGGGCCGGAAG577     AsnGluGluPheLysSerMetSerGlyThrIleGlnLeuGlyArgLys     175180185     CTTATCACAAAATACAATCGCCGGGAGCTGACGGACAAGCTTCTCATC625     LeuIleThrLysTyrAsnArgArgGluLeuThrAspLysLeuLeuIle     190195200205     TTCCTTGCGCTACGCCTGTTTCTTGCTACGGTCCTCTATATTGTGAAA673     PheLeuAlaLeuArgLeuPheLeuAlaThrValLeuTyrIleValLys     210215220     AAGCGGCTCTTTCCATTTTTGTGAGATCCCAAAGGTGCCAGTTCTGGCCCT724     LysArgLeuPheProPheLeu     225     TTCAGCTCCTGTTTCAGGATCTGTCCTGGTTCCTGAGCTCTAGGCTGCTAAGCTGAGCCA784     CACACCCCTCCGTTTTGCACCAGTTGCCTGCAGGTTGGATGGAACACAGTGCCCCACTTT844     TCTGCAAGTAGCTGGCTTGTAAAGGGTGAACAGAGCCATGGGAGGAAGGTCTGGCATTGG904     GATGCCGCCCTGGGGACATACGAACCGCCTCCTTCCACCATTGTGCACTATGGGAGGCCG964     CTGCTGCGTGGAGCACTTAAAGTCCAGCCTCCAGGACCGGATGCCCCTCCTGTCTCCCGC1024     TCCCATCGTGCCCTTAAATGCCAGATCTGGTGGAGGGAAGAGAGAAGAGGTAGGAAGAAA1084     GGTGATGAAAACTCCTG1101     (2) INFORMATION FOR SEQ ID NO:21:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 228 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:     MetAlaAlaProGlnAspValHisValArgIleCysAsnGlnGluIle     151015     ValLysPheAspLeuGluValLysAlaLeuIleGlnAspIleArgAsp     202530     CysSerGlyProLeuSerAlaLeuThrGluLeuAsnThrLysValLys     354045     GluLysPheGlnGlnLeuArgHisArgIleGlnAspLeuGluGlnLeu     505560     AlaLysGluGlnAspLysGluSerGluLysGlnLeuLeuLeuGlnGlu     65707580     ValGluAsnHisLysLysGlnMetLeuSerAsnGlnAlaSerTrpArg     859095     LysAlaAsnLeuThrCysLysIleAlaIleAspAsnLeuGluLysAla     100105110     GluLeuLeuGlnGlyGlyAspLeuLeuArgGlnArgLysThrThrLys     115120125     GluSerLeuAlaGlnThrSerSerThrIleThrGluSerLeuMetGly     130135140     IleSerArgMetMetAlaGlnGlnValGlnGlnSerGluGluAlaMet     145150155160     GlnSerLeuValThrSerSerArgThrIleLeuAspAlaAsnGluGlu     165170175     PheLysSerMetSerGlyThrIleGlnLeuGlyArgLysLeuIleThr     180185190     LysTyrAsnArgArgGluLeuThrAspLysLeuLeuIlePheLeuAla     195200205     LeuArgLeuPheLeuAlaThrValLeuTyrIleValLysLysArgLeu     210215220     PheProPheLeu     225     (2) INFORMATION FOR SEQ ID NO:22:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1386 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 214..1155     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:     CTGCGGCCGGGGGATTGGGCCGGGGTCTCCACCGCCGACCGAGGGGAGCGGCGTCCGCTC60     GGCCCTGCTTTTTGCGACCTGCCGTCAGCCCCACGTCGCCGGCCTGGAGGGGCGAAGAGG120     ACGAGGGGCGACGAAGGCCCAAGGCTTCCTCCGGGGACATTGGCTCCCTGGATTATCAAG180     CAGTTTGTAGTTGACATTGAATCCAGGCTGAGGATGGAAGGTGTGGAACTTAAA234     MetGluGlyValGluLeuLys     230235     GAAGAATGGCAAGATGAAGATTTTCCGATACCTTTACCAGAAGATGAT282     GluGluTrpGlnAspGluAspPheProIleProLeuProGluAspAsp     240245250     AGTATTGAAGCAGATATACTAGCTATAACTGGACCAGAGGACCAGCCT330     SerIleGluAlaAspIleLeuAlaIleThrGlyProGluAspGlnPro     255260265     GGCTCACTAGAAGTTAATGGAAATAAAGTGAGAAAGAAACTAATGGCT378     GlySerLeuGluValAsnGlyAsnLysValArgLysLysLeuMetAla     270275280     CCAGACATTAGCCTGACACTGGATCCTAGTGATGGCTCTGTATTGTCA426     ProAspIleSerLeuThrLeuAspProSerAspGlySerValLeuSer     285290295     GATGATTTGGATGAAAGTGGGGAGATTGACTTAGATGGCTTAGACACA474     AspAspLeuAspGluSerGlyGluIleAspLeuAspGlyLeuAspThr     300305310315     CCGTCAGAGAATAGTAATGAGTTTGAGTGGGAAGATGATCTTCCAAAA522     ProSerGluAsnSerAsnGluPheGluTrpGluAspAspLeuProLys     320325330     CCCAAGACTACTGAAGTAATTAGGAAAGGCTCAATTACTGAATACACA570     ProLysThrThrGluValIleArgLysGlySerIleThrGluTyrThr     335340345     GCAGCAGAGGAAAAAGAAGATGGACGACGCTGGCGTATGTTCAGGATT618     AlaAlaGluGluLysGluAspGlyArgArgTrpArgMetPheArgIle     350355360     GGAGAACAGGACCACAGGGTTGATATGAAGGCAATTGAACCCTATAAA666     GlyGluGlnAspHisArgValAspMetLysAlaIleGluProTyrLys     365370375     AAAGTTATCAGCCATGGGGGATATTATGGGGATGGATTAAATGCCATT714     LysValIleSerHisGlyGlyTyrTyrGlyAspGlyLeuAsnAlaIle     380385390395     GTTGTATTTGCTGTCTGTTTCATGCCTGAAAGTAGTCAGCCTAACTAT762     ValValPheAlaValCysPheMetProGluSerSerGlnProAsnTyr     400405410     AGATACCTGATGGACAATCTTTTTAAATATGTTATTGGCACTTTGGAG810     ArgTyrLeuMetAspAsnLeuPheLysTyrValIleGlyThrLeuGlu     415420425     CTATTAGTAGCAGAAAACTACATGATAGTTTATTTAAATGGTGCAACA858     LeuLeuValAlaGluAsnTyrMetIleValTyrLeuAsnGlyAlaThr     430435440     ACTCGAAGAAAAATGCCCAGTCTGGGATGGCTCAGGAAATGTTATCAG906     ThrArgArgLysMetProSerLeuGlyTrpLeuArgLysCysTyrGln     445450455     CAAATTGATAGAAGGTTACGGAAAAATCTAAAATCCCTAATCATTGTA954     GlnIleAspArgArgLeuArgLysAsnLeuLysSerLeuIleIleVal     460465470475     CATCCTTCTTGGTTTATCAGAACACTTCTGGCTGTTACAAGACCATTT1002     HisProSerTrpPheIleArgThrLeuLeuAlaValThrArgProPhe     480485490     ATTAGCTCGAAATTCAGCCAAAAAATTAGATACGTGTTTAATTTGGCA1050     IleSerSerLysPheSerGlnLysIleArgTyrValPheAsnLeuAla     495500505     GAACTAGCAGAACTTGTCCCCATGGAATACGTTGGCATACCAGAATGC1098     GluLeuAlaGluLeuValProMetGluTyrValGlyIleProGluCys     510515520     ATAAAACAAGTTGATCAAGAACTTAATGGAAAACAAGATGAACCGAAA1146     IleLysGlnValAspGlnGluLeuAsnGlyLysGlnAspGluProLys     525530535     AATGAACAGTAAGTTTGGCATCTAGTCCAAACAAGACTGAAGAATGTGC1195     AsnGluGln     540     TGATGGAGCAGTGCTGTTTCTGCATTCATAATGCATTTATTGGCCATATTTTTATGTAAC1255     CTGTTACAAAATAGACTTGACTTTTTCATAATGGACTTTTGTATTATACAAGGGACTGTT1315     CACTGCTGTACTGGTTTGCAAATTTCTTGAATTTAGCTCTTTATGCTAACTGTATTATTA1375     TCATTTTATAT1386     (2) INFORMATION FOR SEQ ID NO:23:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 314 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:     MetGluGlyValGluLeuLysGluGluTrpGlnAspGluAspPhePro     151015     IleProLeuProGluAspAspSerIleGluAlaAspIleLeuAlaIle     202530     ThrGlyProGluAspGlnProGlySerLeuGluValAsnGlyAsnLys     354045     ValArgLysLysLeuMetAlaProAspIleSerLeuThrLeuAspPro     505560     SerAspGlySerValLeuSerAspAspLeuAspGluSerGlyGluIle     65707580     AspLeuAspGlyLeuAspThrProSerGluAsnSerAsnGluPheGlu     859095     TrpGluAspAspLeuProLysProLysThrThrGluValIleArgLys     100105110     GlySerIleThrGluTyrThrAlaAlaGluGluLysGluAspGlyArg     115120125     ArgTrpArgMetPheArgIleGlyGluGlnAspHisArgValAspMet     130135140     LysAlaIleGluProTyrLysLysValIleSerHisGlyGlyTyrTyr     145150155160     GlyAspGlyLeuAsnAlaIleValValPheAlaValCysPheMetPro     165170175     GluSerSerGlnProAsnTyrArgTyrLeuMetAspAsnLeuPheLys     180185190     TyrValIleGlyThrLeuGluLeuLeuValAlaGluAsnTyrMetIle     195200205     ValTyrLeuAsnGlyAlaThrThrArgArgLysMetProSerLeuGly     210215220     TrpLeuArgLysCysTyrGlnGlnIleAspArgArgLeuArgLysAsn     225230235240     LeuLysSerLeuIleIleValHisProSerTrpPheIleArgThrLeu     245250255     LeuAlaValThrArgProPheIleSerSerLysPheSerGlnLysIle     260265270     ArgTyrValPheAsnLeuAlaGluLeuAlaGluLeuValProMetGlu     275280285     TyrValGlyIleProGluCysIleLysGlnValAspGlnGluLeuAsn     290295300     GlyLysGlnAspGluProLysAsnGluGln     305310     (2) INFORMATION FOR SEQ ID NO:24:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 826 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 109..690     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:     AAAGAGATCTGGAATTCGGATCCTCGAGGCCACGAAGGCCACCGCCCGCAGCTGAAGCAC60     ATCCGCAGCCCGGCGCGACTCCGATCGCCGCAGTTGCCCTCTGGCGCCATGTCCGAG117     MetSerGlu     315     AACGGAGCGCCCGGGATGCAGGAGGAGAGCCTGCAGGGCTCCTGGGTA165     AsnGlyAlaProGlyMetGlnGluGluSerLeuGlnGlySerTrpVal     320325330     GAACTGCACTTCAGCAATAATGGGAACGGGGGCAGCGTTCCAGCCTCG213     GluLeuHisPheSerAsnAsnGlyAsnGlyGlySerValProAlaSer     335340345     GTTTCTATTTATAATGGAGACATGGAAAAAATACTGCTGGACGCACAG261     ValSerIleTyrAsnGlyAspMetGluLysIleLeuLeuAspAlaGln     350355360365     CATGAGTCTGGACGGAGTAGCTCCAAGAGCTCTCACTGTGACAGCCCA309     HisGluSerGlyArgSerSerSerLysSerSerHisCysAspSerPro     370375380     CCTCGCTCGCAGACACCACAAGATACCAACAGGGCTTCTGAAACAGAT357     ProArgSerGlnThrProGlnAspThrAsnArgAlaSerGluThrAsp     385390395     ACCCATAGCATTGGAGAGAAAAACAGCTCACAGTCTGAGGAAGATGAT405     ThrHisSerIleGlyGluLysAsnSerSerGlnSerGluGluAspAsp     400405410     ATTGAAAGAAGGAAAGAAGTTGAAAGCATCTTGAAGAAAAACTCAGAT453     IleGluArgArgLysGluValGluSerIleLeuLysLysAsnSerAsp     415420425     TGGATATGGGATTGGTCAAGTCGGCCGGAAAATATTCCCCCCAAGGAG501     TrpIleTrpAspTrpSerSerArgProGluAsnIleProProLysGlu     430435440445     TTCCTCTTTAAACACCCGAAGCGCACGGCCACCCTCAGCATGAGGAAC549     PheLeuPheLysHisProLysArgThrAlaThrLeuSerMetArgAsn     450455460     ACGAGCGTCATGAAGAAAGGGGGCATATTCTCTGCAGAATTTCTGAAA597     ThrSerValMetLysLysGlyGlyIlePheSerAlaGluPheLeuLys     465470475     GTTTTCCTTCCATCTCTGCTGCTCTCTCATTTGCTGGCCATCGGATTG645     ValPheLeuProSerLeuLeuLeuSerHisLeuLeuAlaIleGlyLeu     480485490     GGGATCTATATTGGAAGGCGTCTGACAACCTCCACCAGCACCTTT690     GlyIleTyrIleGlyArgArgLeuThrThrSerThrSerThrPhe     495500505     TGATGAAGAACTGGAGTCTGACTTGGTTCGTTAGTGGATTACTTCTGAGCTTGCAACATA750     GCTCACTGAAGAGCTGTTAGATCCTGGGCCTTCGTGGCTCGAGAGACTAGAATCGCAGAT810     ACGAAAACCCCGCAGC826     (2) INFORMATION FOR SEQ ID NO:25:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 194 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:     MetSerGluAsnGlyAlaProGlyMetGlnGluGluSerLeuGlnGly     151015     SerTrpValGluLeuHisPheSerAsnAsnGlyAsnGlyGlySerVal     202530     ProAlaSerValSerIleTyrAsnGlyAspMetGluLysIleLeuLeu     354045     AspAlaGlnHisGluSerGlyArgSerSerSerLysSerSerHisCys     505560     AspSerProProArgSerGlnThrProGlnAspThrAsnArgAlaSer     65707580     GluThrAspThrHisSerIleGlyGluLysAsnSerSerGlnSerGlu     859095     GluAspAspIleGluArgArgLysGluValGluSerIleLeuLysLys     100105110     AsnSerAspTrpIleTrpAspTrpSerSerArgProGluAsnIlePro     115120125     ProLysGluPheLeuPheLysHisProLysArgThrAlaThrLeuSer     130135140     MetArgAsnThrSerValMetLysLysGlyGlyIlePheSerAlaGlu     145150155160     PheLeuLysValPheLeuProSerLeuLeuLeuSerHisLeuLeuAla     165170175     IleGlyLeuGlyIleTyrIleGlyArgArgLeuThrThrSerThrSer     180185190     ThrPhe     (2) INFORMATION FOR SEQ ID NO:26:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 946 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 26..505     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:     CAGCATCGCCGCCGCCAGAGGAGAAATGTCTGAAGTAAGACCCCTCTCCAGA52     MetSerGluValArgProLeuSerArg     195200     GACATCTTGATGGAGACCCTCCTGTATGAGCAGCTCCTGGAACCCCCG100     AspIleLeuMetGluThrLeuLeuTyrGluGlnLeuLeuGluProPro     205210215     ACCATGGAGGTTCTTGGCATGACTGACTCTGAAGAGGACCTGGACCCT148     ThrMetGluValLeuGlyMetThrAspSerGluGluAspLeuAspPro     220225230235     ATGGAGGACTTCGATTCTTTGGAATGCATGGAGGGCAGTGACGCATTG196     MetGluAspPheAspSerLeuGluCysMetGluGlySerAspAlaLeu     240245250     GCCCTGCGGCTGGCCTGCATCGGGGACGAGATGGACGTGAGCCTCAGG244     AlaLeuArgLeuAlaCysIleGlyAspGluMetAspValSerLeuArg     255260265     GCCCCGCGCCTGGCCCAGCTCTCCGAGGTGGCCATGCACAGCCTGGGT292     AlaProArgLeuAlaGlnLeuSerGluValAlaMetHisSerLeuGly     270275280     CTGGCTTTCATCTACGACCAGACTGAGGACATCAGGGATGTTCTTAGA340     LeuAlaPheIleTyrAspGlnThrGluAspIleArgAspValLeuArg     285290295     AGTTTCATGGACGGTTTCACCACACTTAAGGAGAACATAATGAGGTTC388     SerPheMetAspGlyPheThrThrLeuLysGluAsnIleMetArgPhe     300305310315     TGGAGATCCCCGAACCCCGGGTCCTGGGTGTCCTGCGAACAGGTGCTG436     TrpArgSerProAsnProGlySerTrpValSerCysGluGlnValLeu     320325330     CTGGCGCTGCTGCTGCTGCTGGCGCTGCTGCTGCCGCTGCTCAGCGGG484     LeuAlaLeuLeuLeuLeuLeuAlaLeuLeuLeuProLeuLeuSerGly     335340345     GGCCTGCACCTGCTGCTCAAGTGAGGCCCCGGCGGCTCAGGGCGTGGCTGG535     GlyLeuHisLeuLeuLeuLys     350     CCCCACCCCCATGACCACTGCCCTGGAGGTGGCGGCCTGCTGCTGTTATCTTTTTAACTG595     TTTTCTCATGATGCCTTTTTATATTTAAACCCCGAGATAGTGCTGGAACACTGCTGAGGT655     TTTATACTCAGGTTTTTTGTTTTTTTTTTATTCCAGTTTTCGTTTTTTCTAAAAGATGAA715     TTCCTATGGCTCTGCAATTGTCACCGGTTAACTGTGGCCTGTGCCCAGGAAGAGCCATTC775     ACTCCTGCCCCTGCCCACACGGCAGGTAGCAGGGGGAGTGCTGGTCACACCCCTGTGTGA835     TATGTGATGCCCTCGGCAAAGAATCTACTGGAATAGATTCCGAGGAGCAGGAGTGCTCAA895     TAAAATGTTGGTTTCCAGCAAAAAAAAAAAAAAGGCCTTCGTGGCCTCGAG946     (2) INFORMATION FOR SEQ ID NO:27:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 160 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:     MetSerGluValArgProLeuSerArgAspIleLeuMetGluThrLeu     151015     LeuTyrGluGlnLeuLeuGluProProThrMetGluValLeuGlyMet     202530     ThrAspSerGluGluAspLeuAspProMetGluAspPheAspSerLeu     354045     GluCysMetGluGlySerAspAlaLeuAlaLeuArgLeuAlaCysIle     505560     GlyAspGluMetAspValSerLeuArgAlaProArgLeuAlaGlnLeu     65707580     SerGluValAlaMetHisSerLeuGlyLeuAlaPheIleTyrAspGln     859095     ThrGluAspIleArgAspValLeuArgSerPheMetAspGlyPheThr     100105110     ThrLeuLysGluAsnIleMetArgPheTrpArgSerProAsnProGly     115120125     SerTrpValSerCysGluGlnValLeuLeuAlaLeuLeuLeuLeuLeu     130135140     AlaLeuLeuLeuProLeuLeuSerGlyGlyLeuHisLeuLeuLeuLys     145150155160     (2) INFORMATION FOR SEQ ID NO:28:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 981 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..777     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:     ATGGACGCCTGGGTCCGCTTCAGTGCTCAGAGCCAAGCCCGGGAGCGG48     MetAspAlaTrpValArgPheSerAlaGlnSerGlnAlaArgGluArg     165170175     CTGTGTAGGGCCGCCCAGTATGCTTGCTCTCTTCTTGGCCATGCGCTG96     LeuCysArgAlaAlaGlnTyrAlaCysSerLeuLeuGlyHisAlaLeu     180185190     CAGAGGCATGGAGCCAGTCCTGAGTTACAGAAACAGATTCGACAACTG144     GlnArgHisGlyAlaSerProGluLeuGlnLysGlnIleArgGlnLeu     195200205     GAGAGCCACCTGAGCCTTGGAAGAAAGCTTCTACGCCTGGGTAACTCA192     GluSerHisLeuSerLeuGlyArgLysLeuLeuArgLeuGlyAsnSer     210215220     GCAGATGCCCTTGAGTCAGCCAAAAGAGCTGTTCACCTATCAGATGTT240     AlaAspAlaLeuGluSerAlaLysArgAlaValHisLeuSerAspVal     225230235240     GTCCTGAGATTCTGCATCACTGTTAGTCACCTCAATCGAGCCTTGTAC288     ValLeuArgPheCysIleThrValSerHisLeuAsnArgAlaLeuTyr     245250255     TTCGCCTGTCACAATGTCCTGTGGGCTGGAAAGTCTGGACTGGCTCCC336     PheAlaCysHisAsnValLeuTrpAlaGlyLysSerGlyLeuAlaPro     260265270     CGTGTGGATCAGGAGAAGTGGGCCCAGCGTTCATTCAGGTACTATTTG384     ArgValAspGlnGluLysTrpAlaGlnArgSerPheArgTyrTyrLeu     275280285     TTTTCCCTCATCATGAATTTGAGCCGTGATGCTTATGAGATTCGCCTA432     PheSerLeuIleMetAsnLeuSerArgAspAlaTyrGluIleArgLeu     290295300     CTGATGGAGCAAGAGTCTTCTGCTTGTAGCCGGCGACTGAAAGGTTCT480     LeuMetGluGlnGluSerSerAlaCysSerArgArgLeuLysGlySer     305310315320     GGAGGAGGAGTCCCAGGAGGAAGTGAAACTGGGGGACTTGGGGGACCA528     GlyGlyGlyValProGlyGlySerGluThrGlyGlyLeuGlyGlyPro     325330335     GGGACTCCAGGAGGAGGTCTGCCCCAACTGGCTCTGAAACTTCGGCTG576     GlyThrProGlyGlyGlyLeuProGlnLeuAlaLeuLysLeuArgLeu     340345350     CAAGTCCTGCTCCTGGCTCGAGTCCTTAGAGGTCATCCCCCACTTCTG624     GlnValLeuLeuLeuAlaArgValLeuArgGlyHisProProLeuLeu     355360365     CTAGACGTGGTCAGAAATGCCTGTGATCTCTTCATTCCTCTGGACAAA672     LeuAspValValArgAsnAlaCysAspLeuPheIleProLeuAspLys     370375380     CTAGCGCTCTGGCGCTGTGGCCCTGGGATTGTGGGGCTTTGTGGCCTC720     LeuAlaLeuTrpArgCysGlyProGlyIleValGlyLeuCysGlyLeu     385390395400     GTGTCCTCCATCCTGTCTATTCTCACCCTAATCTATCCCTGGCTACGA768     ValSerSerIleLeuSerIleLeuThrLeuIleTyrProTrpLeuArg     405410415     CTCAAGCCCTGACTTCCGGTACAGGATAAGGAGGGGACCTGAATTGGTG817     LeuLysPro     AGATGGAATCTTAGATCGTCCCCATGTGCCAGCCTCATTCGAATTCTACTCTTTGGTTAA877     AGTTAGAAATTCAGAGATTTAGGGGTGGAGGAGGAAGAGCTTTGGGGAAGATGAGGTAAG937     GAAAGATGACTCGTGAAGTTAATAGGATGTCTCTAATTTCTAGA981     (2) INFORMATION FOR SEQ ID NO:29:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 259 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:     MetAspAlaTrpValArgPheSerAlaGlnSerGlnAlaArgGluArg     151015     LeuCysArgAlaAlaGlnTyrAlaCysSerLeuLeuGlyHisAlaLeu     202530     GlnArgHisGlyAlaSerProGluLeuGlnLysGlnIleArgGlnLeu     354045     GluSerHisLeuSerLeuGlyArgLysLeuLeuArgLeuGlyAsnSer     505560     AlaAspAlaLeuGluSerAlaLysArgAlaValHisLeuSerAspVal     65707580     ValLeuArgPheCysIleThrValSerHisLeuAsnArgAlaLeuTyr     859095     PheAlaCysHisAsnValLeuTrpAlaGlyLysSerGlyLeuAlaPro     100105110     ArgValAspGlnGluLysTrpAlaGlnArgSerPheArgTyrTyrLeu     115120125     PheSerLeuIleMetAsnLeuSerArgAspAlaTyrGluIleArgLeu     130135140     LeuMetGluGlnGluSerSerAlaCysSerArgArgLeuLysGlySer     145150155160     GlyGlyGlyValProGlyGlySerGluThrGlyGlyLeuGlyGlyPro     165170175     GlyThrProGlyGlyGlyLeuProGlnLeuAlaLeuLysLeuArgLeu     180185190     GlnValLeuLeuLeuAlaArgValLeuArgGlyHisProProLeuLeu     195200205     LeuAspValValArgAsnAlaCysAspLeuPheIleProLeuAspLys     210215220     LeuAlaLeuTrpArgCysGlyProGlyIleValGlyLeuCysGlyLeu     225230235240     ValSerSerIleLeuSerIleLeuThrLeuIleTyrProTrpLeuArg     245250255     LeuLysPro     (2) INFORMATION FOR SEQ ID NO:30:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 2166 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..1503     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:     AGTCAAGTCGAGTTTCTTTGCATGTCACTGGCCAATCCTCTTCCAACA48     SerGlnValGluPheLeuCysMetSerLeuAlaAsnProLeuProThr     260265270275     AATTACACGTGGTACCACAATGGGAAAGAAATGCAGGGAAGGACAGAG96     AsnTyrThrTrpTyrHisAsnGlyLysGluMetGlnGlyArgThrGlu     280285290     GAGAAAGTCCACATCCCAAAGATCCTCCCCTGGCACGCTGGGACTTAT144     GluLysValHisIleProLysIleLeuProTrpHisAlaGlyThrTyr     295300305     TCCTGTGTGGCAGAAAACATTCTTGGTACTGGACAGAGGGGCCCGGGA192     SerCysValAlaGluAsnIleLeuGlyThrGlyGlnArgGlyProGly     310315320     GCTGAGCTGGATGTCCAGTATCCTCCCAAGAAGGTGACCACAGTGATT240     AlaGluLeuAspValGlnTyrProProLysLysValThrThrValIle     325330335     CAAAACCCCATGCCGATTCGAGAAGGAGACACAGTGACCCTTTCCTGT288     GlnAsnProMetProIleArgGluGlyAspThrValThrLeuSerCys     340345350355     AACTACAATTCCAGTAACCCCAGTGTTACCCGGTATGAATGGAAACCC336     AsnTyrAsnSerSerAsnProSerValThrArgTyrGluTrpLysPro     360365370     CATGGCGCCTGGGAGGAGCCATCGCTTGGGGTGCTGAAGATCCAAAAC384     HisGlyAlaTrpGluGluProSerLeuGlyValLeuLysIleGlnAsn     375380385     GTTGGCTGGGACAACACAACCATCGCCTGCGCACGTTGTAATAGTTGG432     ValGlyTrpAspAsnThrThrIleAlaCysAlaArgCysAsnSerTrp     390395400     TGCTCGTGGGCCTCCCCTGTCGCCCTGAATGTCCAGTATGCCCCCCGA480     CysSerTrpAlaSerProValAlaLeuAsnValGlnTyrAlaProArg     405410415     GACGTGAGGGTCCGGAAAATCAAGCCCCTTTCCGAGATTCACTCTGGA528     AspValArgValArgLysIleLysProLeuSerGluIleHisSerGly     420425430435     AACTCGGTCAGCCTCCAATGTGACTTCTCAAGCAGCCACCCCAAAGAA576     AsnSerValSerLeuGlnCysAspPheSerSerSerHisProLysGlu     440445450     GTCCAGTTCTTCTGGGAGAAAAATGGCAGGCTTCTGGGGAAAGAAAGC624     ValGlnPhePheTrpGluLysAsnGlyArgLeuLeuGlyLysGluSer     455460465     CAGCTGAATTTTGACTCCATCTCCCCAGAAGATGCTGGGAGTTACAGC672     GlnLeuAsnPheAspSerIleSerProGluAspAlaGlySerTyrSer     470475480     TGCTGGGTGAACAACTCCATAGGACAGACAGCGTCCAAGGCCTGGACA720     CysTrpValAsnAsnSerIleGlyGlnThrAlaSerLysAlaTrpThr     485490495     CTTGAAGTGCTGTATGCACCCAGGAGGCTGCGTGTGTCCATGAGCCCG768     LeuGluValLeuTyrAlaProArgArgLeuArgValSerMetSerPro     500505510515     GGGGACCAAGTGATGGAGGGGAAGAGTGCAACCCTGACCTGTGAGAGT816     GlyAspGlnValMetGluGlyLysSerAlaThrLeuThrCysGluSer     520525530     GACGCCAACCCTCCCGTCTCCCACTACACCTGGTTTGACTGGAATAAC864     AspAlaAsnProProValSerHisTyrThrTrpPheAspTrpAsnAsn     535540545     CAAAGCCTCCCCCACCACAGCCAGAAGCTGAGATTGGAGCCGGTGAAG912     GlnSerLeuProHisHisSerGlnLysLeuArgLeuGluProValLys     550555560     GTCCAGCACTCGGGTGCCTACTGGTGCCAGGGGACCAACAGTGTGGGC960     ValGlnHisSerGlyAlaTyrTrpCysGlnGlyThrAsnSerValGly     565570575     AAGGGCCGTTCGCCTCTCAGCACCCTTACTGTCTACTATAGCCCGGAG1008     LysGlyArgSerProLeuSerThrLeuThrValTyrTyrSerProGlu     580585590595     ACCATCGGCAGGCGAGTGGCTGTGGGACTCGGGTCCTGCCTCGCCATC1056     ThrIleGlyArgArgValAlaValGlyLeuGlySerCysLeuAlaIle     600605610     CTCATCCTGGCAATCTGTGGGCTCAAGCTCCAGCGACGTTGGAAGAGG1104     LeuIleLeuAlaIleCysGlyLeuLysLeuGlnArgArgTrpLysArg     615620625     ACACAGAGCCAGCAGGGGCTTCAGGAGAATTCCAGCGGCCAGAGCTTC1152     ThrGlnSerGlnGlnGlyLeuGlnGluAsnSerSerGlyGlnSerPhe     630635640     TTTGTGAGGAATAAAAAGGTTAGAAGGGCCCCCCTCTCTGAAGGCCCC1200     PheValArgAsnLysLysValArgArgAlaProLeuSerGluGlyPro     645650655     CACTCCCTGGGATGCTACAATCCAATGATGGAAGATGGCATTAGCTAC1248     HisSerLeuGlyCysTyrAsnProMetMetGluAspGlyIleSerTyr     660665670675     ACCACCCTGCGCTTTCCCGAGATGAACATACCACGAACTGGAGATGCA1296     ThrThrLeuArgPheProGluMetAsnIleProArgThrGlyAspAla     680685690     GAGTCCTCAGAGATGCAGAGACCTCCCCGGACCTGCGATGACACGGTC1344     GluSerSerGluMetGlnArgProProArgThrCysAspAspThrVal     695700705     ACTTATTCAGCATTGCACAAGCGCCAAGTGGGCGACTATGAGAACGTC1392     ThrTyrSerAlaLeuHisLysArgGlnValGlyAspTyrGluAsnVal     710715720     ATTCCAGATTTTCCAGAAGATGAGGGGATTCATTACTCAGAGCTGATC1440     IleProAspPheProGluAspGluGlyIleHisTyrSerGluLeuIle     725730735     CAGTTTGGGGTCGGGGAGCGGCCTCAGGCACAAGAAAATGTGGACTAT1488     GlnPheGlyValGlyGluArgProGlnAlaGlnGluAsnValAspTyr     740745750755     GTGATCCTCAAACATTGACACTGGATGGGCTGCAGCAGAGGCACTGGGGGCAGCG1543     ValIleLeuLysHis     760     GGGGCCAGGGAAGTCCCCGAGTTTCCCCAGACACCGCCACATGGCTTCCTCCTGCGTGCA1603     TGTGCGCACACACACACACACACGCACACACACACACACACACTCACTGCGGAGAACCTT1663     GTGCCTGGCTCAGAGCCAGTCTTTTTGGTGAGGGTAACCCCAAACCTCCAAAACTCCTGC1723     CCCTGTTCTCTTCCACTCTCCTTGCTACCCAGAAATCATCTAAATACCTGCCCTGACATG1783     CACACCTCCCCTGCCCCACCAGCCCACTGGCCATCTCCACCCGGAGCTGCTGTGTCCTCT1843     GGATCTGCTCGTCATTTTCCTTCCCTTCTCCATCTCTCTGGCCCTCTACCCCTGATCTGA1903     CATCCCCACTCACGAATATTATGCCCAGTTTCTGCCTCTGAGGGAAAGCCCAGAAAAGGA1963     CAGAAACGAAGTAGAAAGGGGCCCAGTCCTGGCCTGGCTTCTCCTTTGGAAGTGAGGCAT2023     TGCACGGGGAGACGTACGTATCAGCGGCCCCTTGACTCTGGGGACTCCGGGTTTGAGATG2083     GACACACTGGTGTGGATTAACCTGCCAGGGAGACAGAGCTCACAATAAAAATGGCTCAGA2143     TGCCACTTCAAAGAAAAAAAAAA2166     (2) INFORMATION FOR SEQ ID NO:31:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 501 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:     SerGlnValGluPheLeuCysMetSerLeuAlaAsnProLeuProThr     151015     AsnTyrThrTrpTyrHisAsnGlyLysGluMetGlnGlyArgThrGlu     202530     GluLysValHisIleProLysIleLeuProTrpHisAlaGlyThrTyr     354045     SerCysValAlaGluAsnIleLeuGlyThrGlyGlnArgGlyProGly     505560     AlaGluLeuAspValGlnTyrProProLysLysValThrThrValIle     65707580     GlnAsnProMetProIleArgGluGlyAspThrValThrLeuSerCys     859095     AsnTyrAsnSerSerAsnProSerValThrArgTyrGluTrpLysPro     100105110     HisGlyAlaTrpGluGluProSerLeuGlyValLeuLysIleGlnAsn     115120125     ValGlyTrpAspAsnThrThrIleAlaCysAlaArgCysAsnSerTrp     130135140     CysSerTrpAlaSerProValAlaLeuAsnValGlnTyrAlaProArg     145150155160     AspValArgValArgLysIleLysProLeuSerGluIleHisSerGly     165170175     AsnSerValSerLeuGlnCysAspPheSerSerSerHisProLysGlu     180185190     ValGlnPhePheTrpGluLysAsnGlyArgLeuLeuGlyLysGluSer     195200205     GlnLeuAsnPheAspSerIleSerProGluAspAlaGlySerTyrSer     210215220     CysTrpValAsnAsnSerIleGlyGlnThrAlaSerLysAlaTrpThr     225230235240     LeuGluValLeuTyrAlaProArgArgLeuArgValSerMetSerPro     245250255     GlyAspGlnValMetGluGlyLysSerAlaThrLeuThrCysGluSer     260265270     AspAlaAsnProProValSerHisTyrThrTrpPheAspTrpAsnAsn     275280285     GlnSerLeuProHisHisSerGlnLysLeuArgLeuGluProValLys     290295300     ValGlnHisSerGlyAlaTyrTrpCysGlnGlyThrAsnSerValGly     305310315320     LysGlyArgSerProLeuSerThrLeuThrValTyrTyrSerProGlu     325330335     ThrIleGlyArgArgValAlaValGlyLeuGlySerCysLeuAlaIle     340345350     LeuIleLeuAlaIleCysGlyLeuLysLeuGlnArgArgTrpLysArg     355360365     ThrGlnSerGlnGlnGlyLeuGlnGluAsnSerSerGlyGlnSerPhe     370375380     PheValArgAsnLysLysValArgArgAlaProLeuSerGluGlyPro     385390395400     HisSerLeuGlyCysTyrAsnProMetMetGluAspGlyIleSerTyr     405410415     ThrThrLeuArgPheProGluMetAsnIleProArgThrGlyAspAla     420425430     GluSerSerGluMetGlnArgProProArgThrCysAspAspThrVal     435440445     ThrTyrSerAlaLeuHisLysArgGlnValGlyAspTyrGluAsnVal     450455460     IleProAspPheProGluAspGluGlyIleHisTyrSerGluLeuIle     465470475480     GlnPheGlyValGlyGluArgProGlnAlaGlnGluAsnValAspTyr     485490495     ValIleLeuLysHis     500     (2) INFORMATION FOR SEQ ID NO:32:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:     TyrArgGlyAspArgAlaGlu     15     (2) INFORMATION FOR SEQ ID NO:33:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 4 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:     ProGluSerThr     (2) INFORMATION FOR SEQ ID NO:34:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 58 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:     LeuAlaThrAspMetSerCysHisPheGlnGlnValLysThrMetLys     151015     ThrAlaLeuGlnGlnLeuGluArgIleAspLysSerLysAlaLeuSer     202530     LeuLeuLeuHisAlaAlaAspIleSerHisProThrLysGlnTrpSer     354045     TrpHisSerArgTrpThrLysAlaLeuMet     5055     (2) INFORMATION FOR SEQ ID NO:35:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 125 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS:     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:     IleValPheSerAlaCysArgMetProProSerHisGlnLeuAspHis     151015     SerLysLeuLeuGlyTyrLeuLysHisThrLeuAspGlnTyrValGlu     202530     SerAspTyrThrLeuLeuTyrLeuHisHisGlyLeuThrSerAspAsn     354045     LysProSerLeuSerTrpLeuArgAspAlaTyrArgGluPheAspArg     505560     LysTyrLysLysAsnIleLysAlaLeuTyrIleValHisProThrMet     65707580     PheIleLysThrLeuLeuIleLeuPheLysProLeuIleSerPheLys     859095     PheGlyGlnLysIlePheTyrValAsnTyrLeuSerGluLeuSerGlu     100105110     HisValLysLeuGluGlnLeuGlyIleProArgGlnVal     115120125     __________________________________________________________________________ 

What is claimed:
 1. An isolated polypeptide comprising one member selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23, SEQ ID:25, SEQ ID NO:27, and SEQ ID NO:29.
 2. An isolated nucleic acid molecule comprising one member selected from the group consisting of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28.
 3. An isolated polypeptide consisting of one member selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:32.
 4. The polypeptide of claim 3 which is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
 5. An isolated nucleic acid molecule consisting of a nucleotide sequence that encodes one member selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:32.
 6. The nucleic acid molecule of claim 5 that encodes SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
 7. A method for regulating cell death comprising exposing an isolated cell to a cell death regulating amount of a polypeptide comprising one member selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31.
 8. The method of claim 7 which comprises transfecting said cell with a nucleic acid molecule that directs the expression of said polypeptide.
 9. The method of claim 9, wherein said polypeptide comprises SEQ ID NO:27.
 10. The method of claim 8, wherein said nucleic acid molecule comprises one member selected from the group consisting of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30.
 11. The method of claim 9, which comprises transfecting said cell with a nucleic acid molecule comprising SEQ ID NO:26.
 12. A method for neutralizing activity of a molecule selected from the group consisting of the E1B 19 kD protein, the Bcl-2 protein, and the BHRF-1 protein, said method comprising exposing an isolated cell to an activity neutralizing amount of a polypeptide comprising one member selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31.
 13. The method of claim 12, which comprises transfecting said cell with a nucleic acid molecule that directs the expression of said polypeptide.
 14. The method of claim 12 wherein said molecule is Bcl-2.
 15. The method of claim 12, wherein said nucleic acid molecule comprises one member selected from the group consisting of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30.
 16. A method for detecting molecules that bind to at least one of the polypeptides recited in step (b), said method comprising:(a) lysing cells suspected of comprising said molecules to produce a lysate; (b) exposing said lysate to a polypeptide comprising at least one member selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31; and (c) determining the presence of molecule-polypeptide aggregates, thereby detecting a molecule that binds to at least one of the polypeptides recited in step (b).
 17. An isolated nucleic acid molecule consisting of SEQ ID NO:30. 